Human CTLA-4 antibodies and their uses

ABSTRACT

The present invention provides novel human sequence antibodies against human CTLA-4 and methods of treating human diseases, infections and other conditions using these antibodies.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/198,263, filed Aug. 4, 2011, issued as U.S. Pat. No. 8,318,916, whichis a divisional of U.S. patent application Ser. No. 12/564,756, filedSep. 22, 2009, issued as U.S. Pat. No. 8,017,114, which is acontinuation of U.S. patent application Ser. No. 09/948,939, filed Sep.7, 2001, issued as U.S. Pat. No. 7,605,238, which is acontinuation-in-part of U.S. patent application Ser. No. 09/644,668,filed Aug. 24, 2000, issued as U.S. Pat. No. 6,984,720, which claims thepriority to U.S. provisional patent application Ser. No. 60/150,452,filed Aug. 24, 1999, the contents of all of which are herebyincorporated by reference.

SEQUENCE LISTING

The specification further incorporates by references the SequenceListing submitted via EFS on Feb. 5, 2013. The Sequence Listing textfile, identified as 0773750957seglist.txt, is 43,293 bytes and wascreated on Oct. 11, 2012. The Sequence Listing, electronically filed,does not extend beyond the scope of the specification and does notcontain new matter.

FIELD OF THE INVENTION

The present invention relates generally to molecular immunology and thetreatment of human diseases. In particular, it relates to novel humansequence antibodies against human CTLA-4 and methods of treating humandiseases and infections using these antibodies.

BACKGROUND OF THE INVENTION

The vertebrate immune system requires multiple signals to achieveoptimal immune activation; see, e.g., Janeway, Cold Spring Harbor Symp.Quant. Biol. 54:1-14 (1989); Paul William. E., ed. Raven Press, N.Y.,Fundamental Immunology, 4th edition (1998), particularly chapters 12 and13, pages 411 to 478. Interactions between T lymphocytes (T cells) andantigen presenting cells (APC) are essential to the immune response.Levels of many cohesive molecules found on T cells and APC's increaseduring an immune response (Springer et al., A. Rev. Immunol. 5:223-252(1987); Shaw and Shimuzu, Current Opinion in Immunology, Eds. Kindt andLong, 1:92-97 (1988)); and Hemler, Immunology Today 9:109-113 (1988)).Increased levels of these molecules may help explain why activated APC'sare more effective at stimulating antigen-specific T cell proliferationthan are resting APC's (Kaiuchi et al., J. Immunol. 131:109-114 (1983);Kreiger et al., J. Immunol. 135:2937-2945 (1985); McKenzie, J. Immunol.141:2907-2911 (1988); and Hawrylowicz and Unanue, J. Immunol.141:4083-4088 (1988)).

T cell immune response is a complex process that involves cell-cellinteractions (Springer et al., A. Rev. Immunol. 5:223-252 (1987)),particularly between T and accessory cells such as APC's, and productionof soluble immune mediators (cytokines or lymphokines) (Dinarello (1987)New Engl. Jour. Med. 317:940-945; Sallusto (1997) J. Exp. Med.179:1109-1118). This response is regulated by several T-cell surfacereceptors, including the T-cell receptor complex (Weiss (1986) Ann. Rev.Immunol. 4:593-619) and other “accessory” surface molecules (Allison(1994) Curr. Opin. Immunol. 6:414-419; Springer (1987) supra). Many ofthese accessory molecules are naturally occurring cell surfacedifferentiation (CD) antigens defined by the reactivity of monoclonalantibodies on the surface of cells (McMichael, Ed., Leukocyte TypingIII, Oxford Univ. Press, Oxford, N.Y. (1987)).

Early studies suggested that B lymphocyte activation requires twosignals (Bretscher (1970) Science 169:1042-1049) and now it is believedthat all lymphocytes require two signals for their optimal activation,an antigen specific or clonal signal, as well as a second, antigennon-specific signal. (Janeway, supra). Freeman (1989) J. Immunol.143:2714-2722) isolated and sequenced a cDNA clone encoding a B cellactivation antigen recognized by MAb B7 (Freeman (1987) J. Immunol.138:3260). COS cells transfected with this cDNA have been shown to stainby both labeled MAb B7 and MAb BB-1 (Clark (1986) Human Immunol.16:100-113; Yokochi (1981) J. Immunol. 128:823; Freeman et al., (1989)supra; Freeman et al. (1987), supra). In addition, expression of thisantigen has been detected on cells of other lineages, such as monocytes(Freeman et al., supra).

T helper cell (Th) antigenic response requires signals provided by APCs.The first signal is initiated by interaction of the T cell receptorcomplex (Weiss, J. Clin. Invest. 86:1015 (1990)) with antigen presentedin the context of class II major histocompatibility complex (MHC)molecules on the APC (Allen, Immunol. Today 8:270 (1987)). Thisantigen-specific signal is not sufficient to generate a full response,and in the absence of a second signal may actually lead to clonalinactivation or anergy (Schwartz, Science 248:1349 (1990)), Therequirement for a second “costimulatory” signal provided by the MHC hasbeen demonstrated in a number of experimental systems (Schwartz, supra;Weaver and Unanue, Immunol. Today 11:49 (1990)). The molecular nature ofthis second signal is not completely understood, although it is clear insome cases that both soluble molecules such as interleukin (IL)-1(Weaver and Unanue, supra) and membrane receptors involved inintercellular adhesion (Springer, Nature 346:425 (1990)) can providecostimulatory signals.

CD28 antigen, a homodimeric glycoprotein of the immunoglobulinsuperfamily (Aruffo and Seed, Proc. Natl. Acad. Sci. 84:8573-8577(1987)), is an accessory molecule found on most mature human T cells(Damle et al., J. Immunol. 131:2296-2300 (1983)). Current evidencesuggests that this molecule functions in an alternative T cellactivation pathway distinct from that initiated by the T-cell receptorcomplex (June et al., Mol. Cell. Biol. 7:4472-4481 (1987)). Monoclonalantibodies (MAbs) reactive with CD28 antigen can augment T cellresponses initiated by various polyclonal stimuli (reviewed by June etal., supra). These stimulatory effects may result from MAb-inducedcytokine production (Thompson et al., Proc, Natl. Acad. Sci.86:1333-1337 (1989); and Lindsten et al., Science 244:339-343 (1989)) asa consequence of increased mRNA stabilization (Lindsten et al. (1989),supra). Anti-CD28 mAbs can also have inhibitory effects, i.e., they canblock autologous mixed lymphocyte reactions (Damle et al., Proc. Natl.Acad. Sci. 78:5096-6001 (1981)) and activation of antigen-specific Tcell clones (Lesslauer et al., Eur. J. Immunol. 16:1289-1296 (1986)).

Some studies have indicated that CD28 is a counter-receptor for the Bcell activation antigen, BBB-1 (Linsley et al., Proc. Natl. Acad. Sci.USA 87:5031-5035 (1990)). The B7/BB-1 antigen is hereafter referred toas the “B7 antigen”. The B7 ligands are also members of theimmunoglobulin superfamily but have, in contrast to CD28, two Ig domainsin their extracellular region, an N-terminal variable (V)-like domainfollowed by a constant (C)-like domain.

Delivery of a non-specific costimulatory signal to the T cell requiresat least two homologous B7 family members found on APC's, B7-1 (alsocalled B7, B7.1, or CD80) and B7-2 (also called B7.2 or CD86), both ofwhich can deliver costimulatory signals to T cells via CD28.Costimulation through CD28 promotes T cell activation.

Using genetic fusions of the extracellular portions of B7 antigen andCD28 receptor, and Immunoglobulin (Ig) C.gamma.1 (constant region heavychains), interactions between CD28 and B7 antigen have beencharacterized (Linsley et al., J. Exp. Med. 173:721-730 (1991)).Immobilized B7Ig fusion protein, as well as B7 positive CHO cells, havebeen shown to costimulate T cell proliferation.

T cell stimulation with B7 positive CHO cells also specificallystimulates increased levels of transcripts for IL-2. Additional studieshave shown that anti-CD28 MAb inhibited IL-2 production induced incertain T cell leukemia cell lines by cellular interactions with a Bcell leukemia line (Kohno et al., Cell. Immunol. 131-1-10 (1990)).

CD28 has a single extracellular variable region (V)-like domain (Aruffoand Seed, supra). A homologous molecule, CTLA-4 has been identified bydifferential screening of a murine cytolytic-T cell cDNA library (Brunet(1987) Nature 328:267-270).

CTLA-4 is a T cell surface molecule that was originally identified bydifferential screening of a murine cytolytic T cell cDNA library (Brunetet al., Nature 328:267-270 (1987)). CTLA-4 is also a member of theimmunoglobulin (Ig) superfamily; CTLA-4 comprises a single extracellularIg domain. CTLA-4 transcripts have been found in T cell populationshaving cytotoxic activity, suggesting that CTLA-4 might function in thecytolytic response (Brunet et al., supra; Brunet et al., Immunol. Rev.103-21-36 (1988)). Researchers have reported the cloning and mapping ofa gene for the human counterpart of CTLA-4 (Dariavach et al., Eur. J.Immunol. 18:1901-1905 (1988)) to the same chromosomal region (2q33-34)as CD28 (Lafage-Pochitaloff et al., Immunogenetics 31:198-201 (1990)).Sequence comparison between this human CTLA-4 DNA and that encoding CD28proteins reveals significant homology of sequence, with the greatestdegree of homology in the juxtamembrane and cytoplasmic regions (Brunetet al., 1988, supra; Dariavach et al., 1988, supra).

Some studies have suggested that CTLA-4 has an analogous function as asecondary costimulator (Linsley et al., J. Exp. Med. 176:1595-1604(1992); Wu et al., J Exp. Med. 185:1327-1335 (1997) Lindsley, P. et al.U.S. Pat. Nos. 5,977,318; 5,968,510; 5,885,796; and 5,885,579). However,others have reported that CTLA-4 has an opposing role as a dampener of Tcell activation (Krummel (1995) J. Exp. Med. 182:459-465); Krummel etal., Int'l Immunol. 8:519-523 (1996); Chambers et al., Immunity.7:885-895 (1997)). It has been reported that CTLA-4 deficient micesuffer from massive lymphoproliferation (Chambers et al., supra). It hasbeen reported that CTLA-4 blockade augments T cell responses in vitro(Walunas et al., Immunity. 1:405-413 (1994)) and in vivo (Kearney (1995)J. Immunol. 155:1032-1036), exacerbates antitumor immunity (Leach (1996)Science. 271:1734-1736), and enhances an induced autoimmune disease(Luhder (1998) J. Exp. Med. 187:427-432). It has also been reported thatCTLA-4 has an alternative or additional impact on the initial characterof the T cell immune response (Chambers (1997) Curr. Opin. Immunol.9:396-404; Bluestone (1997) J. Immunol. 158:1989-1993; Thompson (1997)Immunity 7:445-450). This is consistent with the observation that someautoimmune patients have autoantibodies to CTLA-4. It is possible thatCTLA-4 blocking antibodies have a pathogenic role in these patients(Matsui (1999) J. Immunol. 162:4328-4335).

Non-human CTLA-4 antibodies have be used in the various studiesdiscussed above. However, one of the major impediments facing thedevelopment of in vivo therapeutic and diagnostic applications forantibodies in humans is the intrinsic immunogenicity of non-humanimmunoglobulins. For example, when immunocompetent human patients areadministered therapeutic doses of rodent monoclonal antibodies, thepatients produce antibodies against the rodent immunoglobulin sequences;these human anti-mouse antibodies (HAMA) neutralize the therapeuticantibodies and can cause acute toxicity. These and other deficiencies inthe previous antibodies are overcome by the provision of humanantibodies to CTLA-4 by the present invention.

SUMMARY OF THE INVENTION

The present invention provides a human sequence antibody thatspecifically binds to human. CTLA-4 and a human sequence antibody thatspecifically binds to human CTLA-4 which is substantially free ofnon-immunoglobulin associated human proteins.

In a related aspect, the invention also provides atherapeutically-effective human sequence antibody that specificallybinds to human CTLA-4. In some embodiments, thetherapeutically-effective human sequence antibody binds to CTLA-4 on thecell surface of normal human T cells. In other embodiments, the T cellsubpopulations marked by CD antigens CD4, CD8, CD25, and CD69 remainstable during and subsequent to the administration of thetherapeutically-effective human sequence antibody. In other embodiments,the therapeutically-effective human sequence antibody binds CTLA-4 onthe cell surface of normal human T cells. In other embodiments, thehuman sequence antibody is well-tolerated in a patient.

Also provided is a composition of polyclonal antibodies comprising aplurality of human sequence antibodies that specifically bind to humanCTLA-4. The composition of polyclonal antibodies can comprise at leastabout 2, 5, 10, 50, 100, 500 or 1000 different human sequence antibodiesthat specifically bind to human CTLA-4.

The invention also provides human sequence antibodies that specificallybind to human CTLA-4 and which block binding of human CTLA-4 to human B7or do not block binding of human CTLA-4 to human B7.

The invention also provides human sequence antibodies that bind to humanCTLA-4 with an equilibrium association constant (Ka) of at least 10⁸M⁻¹. Also provided are human sequence antibodies that bind to humanCTLA-4 with an equilibrium association constant (Ka) of at least 10⁹M⁻¹.

The invention also provides human sequence antibodies that specificallybind to human CTLA-4 that block binding of human CTLA-4 to human B7 byat least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or100%.

The invention also provides human sequence antibodies that specificallybind to human CTLA-4 having an antibody heavy chain of either IgG orIgM. The IgG antibody heavy chain can be IgG1, IgG2, IgG3 or IgG4. Theinvention also provides human sequence antibodies wherein the antibodylight chain is a kappa light chain. The human sequence antibody can beencoded by human IgG heavy chain and human kappa light chain nucleicacids that comprise nucleotide sequences in their variable regions asset forth in SEQ ID NO:2 through SEQ ID NO:23, respectively.

The invention also provides a human sequence antibody wherein the humansequence antibody is encoded by human IgG heavy chain and human kappalight chain nucleic acids that comprise nucleotide sequences in theirvariable regions as set forth in SEQ ID NO:16 and SEQ ID NO:6,respectively.

The invention also provides a human sequence antibody wherein the humansequence antibody is encoded by human IgG heavy chain and human kappalight chain nucleic acids that comprise nucleotide sequences in theirvariable regions as set forth in SEQ ID NO:18 and SEQ ID NO:8,respectively.

The invention also provides a human sequence antibody wherein the humansequence antibody is encoded by human IgG heavy chain and human kappalight chain nucleic acids that comprise nucleotide sequences in theirvariable regions as set forth in SEQ ID NO:22 and SEQ ID NO:12,respectively.

The invention also provides a human sequence antibody wherein the humansequence antibody is encoded by heavy chain and light chain variableregion amino acid sequences as set for the in SEQ ID NO:17 and SEQ IDNO:7, respectively.

The invention provides a human sequence antibody wherein the humansequence antibody is encoded by heavy chain and light chain variableregion amino acid sequences as set for the in SEQ ID NO:19 and SEQ IDNO:9, respectively.

The invention also provides a human sequence antibody wherein the humansequence antibody is encoded by heavy chain and light chain variableregion amino acid sequences as set for the in SEQ ID NO:23 and SEQ IDNO:13, respectively.

The invention provides a human sequence antibody wherein the humansequence antibody is encoded by human IgG heavy chain and human kappalight chain nucleic acids comprising variable heavy and light chainsequences from V gene segments VH 3-303 and VK A-27, respectively.

The invention also provides a human sequence antibody wherein the humansequence antibody is encoded by human IgG heavy chain and human kappalight chain nucleic acids comprising variable heavy and light chainsequences from V gene segments VH 3-33 and VK L-15, respectively.

Some human sequence antibodies of the invention comprise heavy chainCDR1, CDR2, and CDR3 sequences, SYTMH (SEQ ID NO:27), FISYDGNNKYYADSVKG(SEQ ID NO:32) and TGWLGPFDY (SEQ ID NO:37), respectively, and lightchain CDR1, CDR2, and CDR3 sequences, RASQSVGSSYLA (SEQ ID NO:24),GAFSRAT (SEQ ID NO:29), and QQYGSSPWT (SEQ ID NO:35), respectively.

Some human sequence antibodies of the invention comprise heavy chainCDR1, CDR2, and CDR3 sequences, SYTMH (SEQ ID NO:27), FISYDGSNKHYADSVKG(SEQ ID NO:33) and TGWLGPFDY (SEQ ID NO:37), respectively, and lightchain CDR1, CDR2, and CDR3 sequences, RASQSVSSSFLA (SEQ ID NO:25),GASSRAT (SEQ ID NO:30), and QQYGSSPWT (SEQ ID NO:35), respectively.

Other human sequence antibodies of the invention comprise heavy chainCDR1, CDR2, and CDR3 sequences, SYGMH (SEQ ID NO:28), VIWYDGSNKYYADSVKG(SEQ ID NO:34) and APNYIGAFDV (SEQ ID NO:38), respectively, and lightchain CDR1, CDR2, and CDR3 sequences, RASQGISSWLA (SEQ ID NO:26),AASSLQS (SEQ ID NO:31), and QQYNSYPPT (SEQ ID NO:36), respectively.

The invention also provides human sequence antibodies that specificallybind to human CTLA-4, wherein said human sequence antibody is producedby a transgenic non-human animal. The transgenic non-human animal can bea mouse.

The invention also provides a human sequence antibody that specificallybind to human CTLA-4 that is a Fab fragment.

The invention provides a polyvalent complex comprising at least twohuman sequence antibodies each of which specifically binds to humanCTLA-4. The two different antibodies can be linked to each othercovalently or non-covalently.

The invention provides a nucleic acid encoding a heavy chain of a humansequence antibody. The nucleic acid can comprise a nucleotide sequenceas set forth in SEQ ID NO:1.

The invention provides a transgenic non-human animal having a genomecomprising a human sequence heavy chain transgene and a human sequencelight chain transgene, which animal has been immunized with a humanCTLA-4, or a fragment or an analog thereof, whereby the animal expresseshuman sequence antibodies to the human CTLA-4. The transgenic non-humananimal can be a transgenic mouse. The transgenic mouse can comprise HCo7or HCo12.

The invention provides a hybridoma cell line comprising a B cellobtained from a transgenic non-human animal having a genome comprising ahuman sequence heavy chain transgene and a human sequence light chaintransgene, wherein the hybridoma produces a human sequence antibody thatspecifically binds to human CTLA-4. In a related embodiment, thehybridoma secretes a human sequence antibody that specifically bindshuman CTLA-4 or binding fragment thereof, wherein the antibody isselected from the group consisting of: a human sequence antibodycomprising heavy chain heavy chain CDR1, CDR2, and CDR3 sequences, SYTMH(SEQ ID NO:27), FISYDGNNKYYADSVKG (SEQ ID NO:32) and TGWLGPFDY (SEQ IDNO:37), respectively, and light chain CDR1, CDR2, and CDR3 sequences,RASQSVGSSYLA (SEQ ID NO:24), GAFSRAT (SEQ ID NO:29), and QQYGSSPWT (SEQID NO:35), respectively, and heavy chain and light chain variable regionamino acid sequences as set forth in SEQ ID NO:17 and SEQ ID NO:7,respectively; a human sequence antibody comprising heavy chain CDR1,CDR2, and CDR3 sequences, SYTMH (SEQ ID NO:27), FISYDGSNKHYADSVKG (SEQID NO:33) and TGWLGPFDY (SEQ ID NO:37), respectively, and light chainCDR1, CDR2, and CDR3 sequences, RASQSVSSSFLA (SEQ ID NO:25), GASSRAT(SEQ ID NO:30), and QQYGSSPWT (SEQ ID NO:35), respectively, and heavychain and light chain variable region amino acid sequences as set forthin SEQ ID NO:19 and SEQ ID NO:9, respectively; or a human sequenceantibody of claim 1, comprising heavy chain CDR1, CDR2, and CDR3sequences, SYGMH (SEQ ID NO:28), VIWYDGSNKYYADSVKG (SEQ ID NO:34) andAPNYIGAFDV (SEQ ID NO:38), respectively, and light chain CDR1, CDR2, andCDR3 sequences, RASQGISSWLA (SEQ ID NO:26), AASSLQS (SEQ ID NO:31), andQQYNSYPPT (SEQ ID NO:36), respectively, and heavy chain and light chainvariable region amino acid sequences as set forth in SEQ ID NO:23 andSEQ ID NO:13, respectively.

The invention provides a pharmaceutical composition comprising a humansequence antibody that specifically binds to human CTLA-4 and apharmaceutically acceptable carrier. The pharmaceutical composition canfurther comprise an agent effective to induce an immune response againsta target antigen. Also provided are chemotherapeutic agents. Inaddition, antibodies to immunosuppressive molecules are also provided.

The invention provides a method for inducing, augmenting or prolongingan immune response to an antigen in a patient, comprising administeringto the patient an effective dosage of a human sequence antibody thatspecifically binds to human CTLA-4, wherein the antibody blocks bindingof human CTLA-4 to human B7. The antigen can be a tumor antigen, or theantigen can be from a pathogen. The tumor antigen can also betelomerase. The pathogen can be a virus, a bacterium, a fungus or aparasite. The pathogen can also be an HIV. This method can furthercomprise administering the antigen, or a fragment or an analog thereof,to the patient, whereby the antigen in combination with the humansequence antibody induces, augments or prolongs the immune response. Theantigen can be a tumor antigen or a component of an amyloid formation inthe patient, such as a patient suffering from Alzheimer's disease andthe antigen is AB peptide. This method can further compriseadministering a cytokine to the patient.

The invention provides a method of suppressing an immune response in apatient, comprising administering to the patient an effective dosage ofa polyvalent preparation comprising at least two human sequenceantibodies to human CTLA-4 linked to each other. The invention alsoprovides a method of suppressing an immune response in a patient,comprising administering to the patient an effective dosage of apolyclonal preparation comprising at least two human sequence antibodiesto human CTLA-4.

The present invention further provides isolated or recombinant humansequence antibodies and human monoclonal antibodies which specificallybind to human CTLA-4, as well as compositions containing one or acombination of such antibodies. Some of the human sequence antibodies ofthe invention are characterized by binding to human CTLA-4 with highaffinity, and/or by blocking the interaction of human CTLA-4 with itsligand, the human B7-1 and B7-2 molecules. Accordingly, the humansequence antibodies and the human monoclonal antibodies of the inventioncan be used as diagnostic or therapeutic agents in vivo and in vitro.

The human sequence antibodies of the invention can encompass variousantibody isotypes, or mixtures thereof, such as IgG1, IgG2, IgG3, IgG4,IgM, IgA1, IgA2, IgAsec, IgD, and IgE. Typically, they include IgG1(e.g., IgG1k) and IgM isotypes. The human sequence antibodies can befull-length (e.g., an IgG1 or IgG4 antibody) or can include only anantigen-binding portion (e.g., a Fab, F(ab′)2, Fv or a single chain Fvfragment). Some human sequence antibodies are recombinant human sequenceantibodies. Some human sequence antibodies are produced by a hybridomawhich includes a B cell obtained from a transgenic non-human animal,e.g., a transgenic mouse, having a genome comprising a human heavy chaintransgene and a human light chain transgene. The hybridoma can be madeby, e.g., fusing the B cell to an immortalized cell. Some human sequenceantibodies of the invention are produced by hybridomas referred to as4C8, 4E10, 4E10.5, 5A8, 5C4, 5C4.1.3, 5D7, 5D7.1, 5E10, 5E10.12, 5G1,5G1.4, 6A10, 6C9, 6C9.6, 6D9, 6D9.7, 6G4, 7E4, 7E4.4, 7E6, 7118, 8E8,8E8.4, 8F8, 8F8.19, 8H1, 9810, 9A10.1, 9B9, 9C1, 9G5, 105B, 10B5.8,10B9, 10B9.2, 10D1, 10D1.3, 10E11, 10E4, 10E4.5, 11B4, 11D10, 11E4,11E4.1, 11E8, 11F10, 11F11, 11F9, 1101, 11G1.5, 1C7, 1H8.8, 2A7, 2A7.6,2E2, 2E2,7, 2E7, 2E7.2, 2G1, 2G1.2, 3C12, 3E10, 3E10.5, 3E6, 3E6.0,3F10, 4A1, 4B6 and 4B6.12. Suffixes after the decimal point indicatedifferent clonal isolates of the same hybridoma cell lines.

Some human sequence anti-CTLA-4 antibodies of the present invention canbe characterized by one or more of the following properties: a)specificity for human CTLA-4 (specifically binding to human CTLA-4); b)a binding affinity to human CTLA-4 with an equilibrium associationconstant (K_(a)) of at least about 10⁷M⁻¹, or about 10⁹M⁻¹, or about10¹⁰ M⁻¹ to 10¹¹ M⁻¹ or higher; c) a kinetic association constant(k_(a)) of at least about 10³, about 10⁴, or about 10⁵ m⁻¹s⁻¹; and/or,d) a kinetic disassociation constant (k_(d)) of at least about 10³,about 10⁴, or about 10⁵ m⁻¹s⁻¹.

In another aspect, the invention provides nucleic acid moleculesencoding the human sequence antibodies, or antigen-binding portions, ofthe invention. Accordingly, recombinant expression vectors that includethe antibody-encoding nucleic acids of the invention, and host cellstransfected with such vectors, are also encompassed by the invention, asare methods of making the antibodies of the invention by culturing thesehost cells.

In yet another aspect, the invention provides isolated B-cells from atransgenic non-human animal, e.g., a transgenic mouse, which are capableof expressing various isotypes (e.g., IgG, IgA and/or IgM) of humanmonoclonal antibodies that specifically bind to human CTLA-4. Theisolated B cells can be obtained from a transgenic non-human animal,e.g., a transgenic mouse, which has been immunized with a purified orenriched preparation of human CTLA-4 antigen (or antigenic fragmentthereof) and/or cells expressing human CTLA-4. The transgenic non-humananimal, e.g., a transgenic mouse, can have a genome comprising a humanheavy chain transgene and a human light chain transgene. The isolatedB-cells can be immortalized to provide a source (e.g., a hybridoma) ofhuman monoclonal antibodies to human CTLA-4.

Accordingly, the present invention also provides a hybridoma capable ofproducing human monoclonal antibodies that specifically bind to humanCTLA-4. The hybridoma can include a B cell obtained from a transgenicnon-human animal, e.g., a transgenic mouse, having a genome comprising ahuman heavy chain transgene and a human light chain transgene fused toan immortalized cell. The transgenic non-human animal can be immunizedwith a purified or enriched preparation of human CTLA-4 antigen and/orcells expressing human CTLA-4 to generate antibody-producing hybridomas.

In yet another aspect, the invention provides a transgenic non-humananimal, such as a transgenic mouse, which express human monoclonalantibodies (also referred to herein as a “HuMAb-Mouse™”) thatspecifically bind to human CTLA-4. The transgenic non-human animal canbe a transgenic mouse having a genome comprising a human heavy chaintransgene and a human light chain transgene. The transgenic non-humananimal can be immunized with a purified or enriched preparation ofCTLA-4 antigen (or antigenic fragment thereof) and/or cells expressingthe human CTLA-4. The transgenic non-human animal, e.g., the transgenicmouse, can be capable of producing multiple isotypes of human monoclonalantibodies to human CTLA-4 (e.g., IgG, IgA and/or IgM) by undergoingV-D-J recombination and isotype switching. Isotype switching may occurby, e.g., classical or non-classical isotype switching.

In another aspect, the present invention provides methods for producinghuman sequence antibodies and human sequence monoclonal antibodies thatspecifically react with human CTLA-4. Some methods of the inventioninclude immunizing a transgenic non-human animal, e.g., a transgenicmouse, having a genome comprising a human heavy chain transgene and ahuman light chain transgene, with a purified or enriched preparation ofhuman CTLA-4 antigen and/or cells expressing human CTLA-4. B cells(e.g., splenic B cells) of the animal can then be obtained and fusedwith myeloma cells to form immortal, hybridoma cells that secrete humanmonoclonal antibodies against human CTLA-4.

Anti-human CTLA-4 human monoclonal antibodies of the invention, orantigen binding portions thereof (e.g., Fab), can be derivatized orlinked to another functional molecule, e.g., another peptide or protein(e.g., an Fab′ fragment). For example, an antibody or antigen-bindingportion of the invention can be functionally linked (e.g., by chemicalcoupling, genetic fusion, noncovalent association or otherwise) to oneor more other molecular entities. For example, the human sequenceanti-CTLA-4 antibody, or antigen binding fragment thereof, can beconjugated to a therapeutic moiety, e.g., a cytotoxic drug, anenzymatically active toxin, or a fragment thereof, a radioisotope, or asmall molecule anti-cancer drug. The antibodies of the invention canalso be conjugated to cytotoxic pharmaceuticals, e.g., radiolabeled witha cytotoxic agents, such as, e.g., ¹³¹I (e.g., Shen (1997) Cancer 80(12Suppl):2553-2557), copper-67 (e.g., Deshpande (1988) J. Nucl. Med.29:217-225) or, e.g., conjugation to the ribosome inactivating proteingelonin (e.g., Boyle (1996) J. of Immunol. 18:221-230).

In another aspect, the present invention provides compositions, e.g.,pharmaceutical and diagnostic compositions, comprising apharmaceutically acceptable carrier and at least one human monoclonalantibody of the invention, or an antigen-binding portion thereof, whichspecifically binds to human CTLA-4. Some compositions comprise acombination of the human sequence antibodies or antigen-binding portionsthereof, preferably each of which binds to a distinct epitope.Compositions, e.g., pharmaceutical compositions, comprising acombination of at least one human sequence antibodies or at least onehuman monoclonal antibody of the invention, or antigen-binding portionsthereof, and at least one bispecific or multispecific molecule of theinvention, are also within the scope of the invention.

For in vivo methods, the antibody, or antigen-binding portion thereof(or a bispecific or multispecific molecule of the invention), can beadministered to a human subject suffering from a T-cell-related disease,or a disease that can be ameliorated or prevented by augmenting orsuppressing or prolonging an immune response.

Human sequence monoclonal antibody and human sequence antibodycompositions of the invention also can be administered in combinationwith other known therapies, e.g., an anti-cancer therapy. Accordingly,the invention provides a method for treating cancer in a subjectcomprising administering a therapeutically effective amount of apharmaceutical composition of a human sequence antibody together with apharmaceutical carrier to the subject. Some such methods include avaccine. Some such vaccines include a tumor cell vaccine, aGM-CSF-modified tumor cell vaccine, or an antigen-loaded dendritic cellvaccine. In some such methods, the cancer is prostate cancer, melanoma,or epithelial cancer.

Human sequence antibodies to human CTLA-4 can be used in methods oftreatment requiring either stimulation of immune responses orsuppression. The former indication is treated using antibodies thatblock binding of human CTLA-4 to human B7. Diseases amenable totreatment by stimulation, augmentation of prolonging of immune responsesincluding cancer, including cancers of the prostate, kidney or colon,pathogenic infections, diseases associated with auto-antigens, e.g.,amyloidogenic diseases, including Alzheimer's disease, and diseases withinflammatory or allergic components. Immunosuppression is achieved usinga polyvalent preparation comprising at least two different antibodies tohuman CTLA-4 that are linked to each other. Diseases amenable totreatment include graft versus host disease, host versus graft disease,autoimmune diseases and inflammation.

In yet another aspect, the present invention provides a method fordetecting in vitro or in vivo the presence of human CTLA-4 antigen in asample, e.g., for diagnosing a human CTLA-4-related disease. In somemethods, this is achieved by contacting a sample to be tested, alongwith a control sample, with a human sequence antibody or a humanmonoclonal antibody of the invention, or an antigen-binding portionthereof (or a bispecific or multispecific molecule), under conditionsthat allow for formation of a complex between the antibody and humanCTLA-4. Complex formation is then detected (e.g., using an ELISA) inboth samples, and any statistically significant difference in theformation of complexes between the samples is indicative the presence ofhuman CTLA-4 antigen in the test sample.

A further understanding of the nature and advantages of the presentinvention may be realized by reference to the remaining portions of thespecification, the figures and claims.

All publications, figures, GenBank Accession references (sequences),ATCC Deposits, patents and patent applications cited herein are herebyexpressly incorporated by reference for all purposes to the same extentas if each was so individually denoted.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows schematics illustrating the targeted insertion of a neocassette into the Sma I site of the μl exon. FIG. 1A) Schematic diagramof the genomic structure of the μ locus. The filled boxes represent theμ exons; FIG. 1B) Schematic diagram of the CmD targeting vector. Thedotted lines denotes those genomic μ sequences included in theconstruct. Plasmid sequences are not shown; FIG. 1C) Schematic diagramof the targeted locus in which the neo cassette has been inserted intoμl. The box at the lower right shows those RFLP's diagnostic ofhomologous recombination between the targeting construct and the μlocus. The RFLP's were detected by Southern blot hybridization usingprobe A, the 915 bp Sac I fragment is shown in FIG. 1C.

FIGS. 2A-B both show the results of experiments demonstrating thatsoluble human sequence antibodies against human CTLA-4 inhibit thebinding of recombinant soluble human CTLA-4 to cells expressing mouseB7.1, as described in detail, below.

FIG. 3 shows the results of a competitive binding assay to identifyhuman sequence antibodies of the invention that recognizenon-overlapping epitopes on human CTLA-4, as described in detail, below.

FIGS. 4A-B show preliminary nucleotide sequence data for the heavy (FIG.4A; SEQ ID NO:2) and light chain (FIG. 4B; SEQ ID NO:4) fragment of theanti-CTLA-4 antibody 10D1.3.

FIGS. 5 and 5A show the nucleotide sequences of the light chain variableRegions (V_(K)) of Anti-Human CTLA-4 Antibodies. The anti-CTLA-4antibodies 10D1 (SEQ ID NO:6) and 4B6 (SEQ ID NO:8) derived from theV_(K) A-27 germline sequence (SEQ ID NO:4) are depicted at the top ofFIG. 5. The anti-CTLA-4 antibody 1E2 (SEQ ID NO:12) derived from theV_(K) L-15 germline sequence (SEQ ID NO:10) is shown at the bottom ofFIG. 5 and FIG. 5A. The V_(K) sequences of three anti-CTLA-4 antibodiesare aligned with their germline encoded V_(K) gene sequences. Thecomplementary determining residues (CDR) are labeled. Dashes denotesequence identity.

FIGS. 6 and 6A show the nucleotide sequences of the heavy chain variableRegions (V_(H)) of Anti-Human CTLA-4 Antibodies. The anti-CTLA-4antibodies 10D1 (SEQ ID NO:16) and 4B6 (SEQ ID NO:18) derived from theV_(H) 3-30.3 germline sequence (SEQ ID NO:14) are depicted at the top ofFIG. 6. The anti-CTLA-4 antibody 1 E2 (SEQ ID NO:22) derived from theV_(H) 3-33 germline sequence (SEQ ID NO:20) is shown at the bottom ofFIG. 6 and FIG. 6A. The V_(H) sequences of three anti-CTLA-4 antibodiesare aligned with their germline encoded sequences. The complementarydetermining residues (CDR) are labeled. Dashes denote sequence identity.

FIG. 7 shows the predicted amino acid sequences of the light chainVariable Regions of Anti-Human CTLA-4 Antibodies. The predicted aminoacid V_(K) sequences of the anti-CTLA-4 antibodies described in FIG. 5are shown. The anti-CTLA-4 antibodies 10D1 (SEQ ID NO:7) and 4B6 (SEQ IDNO:9) derived from the V_(K) A-27 germline sequence (SEQ ID NO:5) aredepicted at the top of the Figure. The anti-CTLA-4 antibody 1E2 (SEQ IDNO:13) derived from the V_(K) L-15 germline sequence (SEQ ID NO:11) isshown at the bottom of the Figure.

FIG. 8 shows the predicted amino acid sequences of the heavy chainVariable Regions of Anti-Human CTLA-4 Antibodies. The predicted aminoacid V_(H) sequences of the anti-CTLA-4 antibodies described in FIG. 6are shown. The anti-CTLA-4 antibodies 10D1 (SEQ ID NO:17) and 4B6 (SEQID NO:19) derived from the V_(H) 3-30.3 germline sequence (SEQ ID NO:15)are depicted at the top of the Figure. The anti-CTLA-4 antibody 1 E2(SEQ ID NO:23) derived from the V_(H) 3-33 germline sequence (SEQ IDNO:21) is shown at the bottom of the Figure.

FIG. 9 shows the results of binding experiments of MAb 10D1 torecombinant human CTLA-4 by ELISA. MAb 10D1 binds with dose-dependentand saturating kinetics to purified recombinant CTLA-4.

FIG. 10 shows the binding of 10D1 to a CTLA4-expressing T-cell line.These data show that MAb 10D1 binds with dose-dependent and saturatingkinetics to cells expressing CTLA-4.

FIG. 11 shows inhibition of binding of human B7.21 g to CTLA4-expressingT-cells. These data show that MAb 10D1 can efficiently block B7.2binding to CTLA-4 as compared to a control human MAb.

FIG. 12 shows the results for blocking CTLA4-FITC binding to murineB7.1-expressing cells. These data show that MAb 10D1 can efficientlyblock CTLA-4 binding to B7.1 as compared to a control human MAb.

FIGS. 13A-G show competitive ELISAs of anti-CTLA-4 human MAbsdemonstrating epitope group classifications. FIG. 13(A) shows results ofantibody 9A5 competitive ELISA. FIG. 13(B) shows results of antibody 3A4competitive ELISA. FIG. 13(C) shows results of antibody 5A8 competitiveELISA. FIG. 13(D) shows results of antibody 10D1.3 competitive ELISA.FIG. 13(E) shows results of antibody 486.12 competitive ELISA. FIG.13(F) shows results of antibody 147 competitive ELISA. FIG. 13(G) showsresults of antibody BNI 3.1 competitive ELISA.

FIGS. 14A-B show CTLA-4 expression on PHA-stimulated T-cells. ActivatedT cells express low but detectable levels of CTLA-4 at the cell surface,as shown in FIG. 14B, and control T cells do not (FIG. 14A).

FIG. 15 shows the results of MAb 10D1 in Complement Dependent Lysis ofActivated T Cells. No lysis of PHA-activated T cells is observed.

FIG. 16 shows the results of MAb 10D1 in Antibody-Dependent Lysis ofActivated T Cells. No lysis of PHA-activated T cells is observed with10D1 and mononuclear cells.

FIGS. 17A-D show anti-10D1 IgM and IgG responses in cynomolgus monkeysinjected with 10D1 antibody. FIGS. 17A and 17C show anti-10D1 IgMresponse. FIGS. 17B and 17D show anti-10D1 IgG response. No significantantibody response to 10D1 is observed.

FIG. 18 shows prostate specific antigen (PSA) levels in ng/ml in twohuman patients at various time points after infusion of an anti-CTLA4antibody at day 0.

FIGS. 19A-B show the plasma levels of anti-HbsAg antibody in primatestreated with either a HbsAg vaccine in combination with the anti-CTLA4antibody 10D1 or the vaccine in combination with a control IgG1antibody.

FIG. 20 shows the level of antibody responses to a melanoma cell vaccinein primates treated with either the vaccine alone (open circles) or withthe vaccine in combination with the anti-CTLA4 antibody 10D1 (closedcircles).

FIG. 21 shows antigen-specific T cell proliferation in a primatevaccinated with a melanoma cell vaccine in combination with theanti-CTLA4 antibody 10D1.

DETAILED DESCRIPTION

The present invention provides novel antibody-based therapies fortreating and diagnosing diseases characterized by expression,particularly over-expression, or activation of, particularlyoveractivation, of human CTLA-4 and/or related molecules. Therapies ofthe invention employ human sequence antibodies, human sequencemonoclonal antibodies, or antigen-binding portions thereof, which bindto an epitope present on human CTLA-4. These human sequence anti-CTLA-4antibodies can act as functional antagonists (e.g., inhibiting theability of CTLA-4 to bind ligand or to activate the cell, e.g., byinhibiting its ability to transmit a signal to the cell) or agonists(e.g., to simulate the effect of ligand).

The human sequence antibodies of the invention can be produced in anon-human transgenic animal, e.g., a transgenic mouse, capable ofproducing multiple isotypes of human (e.g., monoclonal or polyclonal)antibodies to human CTLA-4 IgG, IgA and/or IgE) by undergoing V-D-Jrecombination and isotype switching. Accordingly, various aspects of theinvention include antibodies and antibody fragments, and pharmaceuticalcompositions thereof, as well as non-human transgenic animals, andB-cells and hybridomas for making such monoclonal antibodies. Methods ofusing the antibodies of the invention to detect a cell expressing humanCTLA-4 or a related, cross-reactive growth factor receptor, or toinhibit growth, differentiation and/or motility of a cell expressinghuman CTLA-4, either in vitro or in vivo, are also encompassed by theinvention.

Except when noted, the terms “patient” or “subject” are usedinterchangeably and refer to mammals such as human patients andnon-human primates, as well as experimental animals such as rabbits,rats, and mice, and other animals.

The term “treating” includes the administration of the compounds oragents of the present invention to prevent or delay the onset of thesymptoms, complications, or biochemical indicia of a disease,alleviating the symptoms or arresting or inhibiting further developmentof the disease, condition, or disorder (e.g., autoimmune disease).Treatment may be prophylactic (to prevent or delay the onset of thedisease, or to prevent the manifestation of clinical or subclinicalsymptoms thereof) or therapeutic suppression or alleviation of symptomsafter the manifestation of the disease.

In general, the phrase “well tolerated” refers to the absence of adversechanges in health status that occur as a result of the treatment andwould affect treatment decisions.

The term “lymphocyte” as used herein has the normal meaning in the art,and refers to any of the mononuclear, nonphagocytic leukocytes, found inthe blood, lymph, and lymphoid tissues, i.e., B and T lymphocytes.

The phrase “subpopulations of T lymphocytes” or “T cell subset(s)”refers to T lymphocytes or T cells characterized by the expression ofparticular cell surface markers (see Barclay, A. N. et al., (eds.),1997, The Leukocyte Antigen Facts Book, 2nd. edition, Academic Press,London, United Kingdom). The term “stable” in reference to T cellsrefers to the fact that the frequency or percentage of a T cell subsetdoes not change over the course or duration of the administration of anagent.

The terms “cytotoxic T lymphocyte-associated antigen-4,” “CTLA-4,”“CTLA4,” “CTLA-4 antigen” and “CD152” (see, e.g., Murata (1999) Am. J.Pathol. 155:453-460) are used interchangeably, and include variants,isoforms, species homologs of human CTLA-4, and analogs having at leastone common epitope with CTLA-4 (see, e.g., Balzano (1992) Int. J. CancerSuppl. 7:28-32).

The complete cDNA sequence of human CTLA-4 has the Genbank accessionnumber L15006. The region of amino acids 1-37 is the leader peptide;38-161 is the extracellular V-like domain; 162-187 is the transmembranedomain; and 188-223 is the cytoplasmic domain. Variants of thenucleotide sequence have been reported, including a G to A transition atposition 49, a C to T transition at position 272, and an A to Gtransition at position 439. The complete DNA sequence of mouse CTLA-4has the EMBL accession number X05719 (Brunet et al. (1987) Nature328:267-270). The region of amino acids 1-35 is the leader peptide.

The complete DNA sequence of human B7-1 (CD80) has the Genbank accessionnumber X60958; the accession number for the mouse sequence is X60958;the accession number for the rat sequence is U05593. The complete cDNAsequence of human B7-2 (CD86) has the Genbank accession number L25259;the accession number for the mouse sequence is L25606.

The genes encoding CD28 have been extensively characterized. The chickenmRNA sequence has the Genbank accession number X67915. The rat mRNAsequence has the Genbank accession number X55288. The human mRNAsequence has the Genbank accession number J02988. The mouse mRNAsequence has the Genbank accession number M34536.

The term “epitope” means a protein determinant capable of specificbinding to an antibody. Epitopes usually consist of chemically activesurface groupings of molecules such as amino acids or sugar side chainsand usually have specific three dimensional structural characteristics,as well as specific charge characteristics. Conformational andnonconformational epitopes are distinguished in that the binding to theformer but not the latter is lost in the presence of denaturingsolvents.

An intact “antibody” comprises at least two heavy (H) chains and twolight (L) chains inter-connected by disulfide bonds. Each heavy chain iscomprised of a heavy chain variable region (abbreviated herein as HCVRor VH) and a heavy chain constant region. The heavy chain constantregion is comprised of three domains, CH1, CH2 and CH3. Each light chainis comprised of a light chain variable region (abbreviated herein asLCVR or VL) and a light chain constant region. The light chain constantregion is comprised of one domain, CL. The VH and VL regions can befurther subdivided into regions of hypervariability, termedcomplementarity determining regions (CDR), interspersed with regionsthat are more conserved, termed framework regions (FR). Each VH and VLis composed of three CDRs and four FRs, arranged from amino-terminus tocarboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. The variable regions of the heavy and light chains contain abinding domain that interacts with an antigen. The constant regions ofthe antibodies may mediate the binding of the immunoglobulin to hosttissues or factors, including various cells of the immune system (e.g.,effector cells) and the first component (Clq) of the classicalcomplement system. The term antibody includes antigen-binding portionsof an intact antibody that retain capacity to bind CTLA-4. Examples ofbinding include (i) a Fab fragment, a monovalent fragment consisting ofthe VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalentfragment comprising two Fab fragments linked by a disulfide bridge atthe hinge region; (iii) a Fd fragment consisting of the VH and CH1domains; (iv) a Fv fragment consisting of the VL and VH domains of asingle arm of an antibody, (v) a dAb fragment (Ward et al., (1989)Nature 341:544-546), which consists of a VH domain; and (vi) an isolatedcomplementarity determining region (CDR). Furthermore, although the twodomains of the Fv fragment, VL and VH, are coded for by separate genes,they can be joined, using recombinant methods, by a synthetic linkerthat enables them to be made as a single protein chain in which the VLand VH regions pair to form monovalent molecules (known as single chainFv (scFv); See, e.g., Bird et al. (1988) Science 242:423-426; and Hustonet al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such singlechain antibodies are included by reference to the term “antibody”Fragments can be prepared by recombinant techniques or enzymatic orchemical cleavage of intact antibodies.

A bispecific antibody has two different binding specificities, see.e.g., U.S. Pat. Nos. 5,922,845 and 5,837,243; Zeilder (1999) J. Immunol.163:1246-1252; Somasundaram (1999) Hum. Antibodies 9:47-54; Keler (1997)Cancer Res. 57:4008-4014. For example, the invention provides bispecificantibodies having one binding site for a cell surface antigen, such ashuman CTLA-4, and a second binding site for an Fe receptor on thesurface of an effector cell. The invention also provides multispecificantibodies, which have at least three binding sites. The term“bispecific antibodies” further includes diabodies. Diabodies arebivalent, bispecific antibodies in which the VH and VL domains areexpressed on a single polypeptide chain, but using a linker that is tooshort to allow for pairing between the two domains on the same chain,thereby forcing the domains to pair with complementary domains ofanother chain and creating two antigen binding sites (See, e.g.,Holliger, P., et al., (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448;Poljak, R. J., et al., (1994) Structure 2:1121-1123).

The term “human sequence antibody” includes antibodies having variableand constant regions (if present) derived from human germlineimmunoglobulin sequences. The human sequence antibodies of the inventionmay include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo).However, the term “human sequence antibody”, as used herein, is notintended to include antibodies in which CDR sequences derived from thegermline of another mammalian species, such as a mouse, have beengrafted onto human framework sequences (i.e., humanized antibodies).

The terms “monoclonal antibody” or “monoclonal antibody composition”refer to a preparation of antibody molecules of single molecularcomposition. A monoclonal antibody composition displays a single bindingspecificity and affinity for a particular epitope. Accordingly, the term“human monoclonal antibody” refers to antibodies displaying a singlebinding specificity which have variable and constant regions (ifpresent) derived from human germline immunoglobulin sequences. In oneembodiment, the human monoclonal antibodies are produced by a hybridomawhich includes a B cell obtained from a transgenic non-human animal,e.g., a transgenic mouse, having a genome comprising a human heavy chaintransgene and a light chain transgene fused to an immortalized cell.

The term “diclonal antibody” refers to a preparation of at least twoantibodies to human CTLA-4. Typically, the different antibodies binddifferent epitopes.

The term “oligoclonal antibody” refers to a preparation of 3 to 100different antibodies to human CTLA-4. Typically, the antibodies in sucha preparation bind to a range of different epitopes.

The term “polyclonal antibody” refers to a preparation of more than 1(two or more) different antibodies to human CTLA-4. Such a preparationincludes antibodies binding to a range of different epitopes.

The invention provides human sequence antibodies to human CTLA-4 whichblock or antagonize signals transduced by the human CTLA-4 receptor.Some of these antibodies can bind to an epitope on human CTLA-4 so as toinhibit CTLA-4 from interacting with a human B7 counterreceptor. Becauseinteraction of human CTLA-4 with human B7 transduces a signal leading toinactivation of T-cells bearing the human CTLA-4 receptor, antagonism ofthe interaction effectively induces, augments or prolongs the activationof T cells bearing the human CTLA-4 receptor, thereby prolonging oraugmenting an immune response. A “blocking antibody” refers to anantibody that reduces the binding of soluble human CTLA-4 tocell-expressed human B7 ligand by at least 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 99% or 99.9% under conditions in which the ratio ofantibody combining site to human CTLA-4 ligand binding site is greaterthan 1:1 and the concentration of antibody is greater than 10⁻⁸ M.

Other antibody preparations, sometimes referred to as multivalentpreparations, bind to human CTLA-4 in such a manner as to crosslinkmultiple human CTLA-4 receptors on the same cell. Cross-linking ofreceptor has the same or similar effect to binding of human CTLA-4 tohuman B7. Thus, cross-linking of receptors effectively agonizes thehuman CTLA-4 response resulting in immunosuppression.

Cross-linking can also be accomplished by combining soluble divalentantibodies having different epitope specificities. These polyclonalantibody preparations comprise at least two pairs of heavy and lightchains binding to different epitopes on human CTLA-4 such that animmunosuppressing signal can be transduced as a result of human CTLA-4crosslinking.

The term “recombinant human antibody” includes all human sequenceantibodies of the invention that are prepared, expressed, created orisolated by recombinant means, such as antibodies isolated from ananimal (e.g., a mouse) that is transgenic for human immunoglobulin genes(described further in Section I, below); antibodies expressed using arecombinant expression vector transfected into a host cell, antibodiesisolated from a recombinant, combinatorial human antibody library, orantibodies prepared, expressed, created or isolated by any other meansthat involves splicing of human immunoglobulin gene sequences to otherDNA sequences. Such recombinant human antibodies have variable andconstant regions (if present) derived from human germline immunoglobulinsequences. Such antibodies can, however, be subjected to in vitromutagenesis (or, when an animal transgenic for human Ig sequences isused, in vivo somatic mutagenesis) and thus the amino acid sequences ofthe VH and VL regions of the recombinant antibodies are sequences that,while derived from and related to human germline VH and VL sequences,may not naturally exist within the human antibody germline repertoire invivo.

A “heterologous antibody” is defined in relation to the transgenicnon-human organism producing such an antibody. This term refers to anantibody having an amino acid sequence or an encoding nucleic acidsequence corresponding to that found in an organism not consisting ofthe transgenic non-human animal, and generally from a species other thanthat of the transgenic non-human animal.

A “heterohybrid antibody” refers to an antibody having a light and heavychains of different organismal origins. For example, an antibody havinga human heavy chain associated with a murine light chain is aheterohybrid antibody. Examples of heterohybrid antibodies includechimeric and humanized antibodies, discussed supra.

The term “substantially pure” or “isolated” means an object species(e.g., an antibody of the invention) has been identified and separatedand/or recovered from a component of its natural environment such thatthe object species is the predominant species present (i.e., on a molarbasis it is more abundant than any other individual species in thecomposition); a “substantially pure” or “isolated” composition alsomeans where the object species comprises at least about 50 percent (on amolar basis) of all macromolecular species present. A substantially pureor isolated composition can also comprise more than about 80 to 90percent by weight of all macromolecular species present in thecomposition. An isolated object species (e.g., antibodies of theinvention) can also be purified to essential homogeneity (contaminantspecies cannot be detected in the composition by conventional detectionmethods) wherein the composition consists essentially of derivatives ofa single macromolecular species. An isolated antibody to human CTLA-4can be substantially free of other antibodies that lack binding to humanCTLA-4 and bind to a different antigen. An isolated antibody thatspecifically binds to an epitope, isoform or variant of human CTLA-4may, however, have cross-reactivity to other related antigens, e.g.,from other species (e.g., CTLA-4 species homologs). Moreover, anisolated antibody of the invention be substantially free of othercellular material (e.g., non-immunoglobulin associated proteins) and/orchemicals.

“Specific binding” refers to antibody binding to a predeterminedantigen. The phrase “specifically (or selectively) binds” to an antibodyrefers to a binding reaction that is determinative of the presence ofthe protein in a heterogeneous population of proteins and otherbiologics. Typically, the antibody binds with an association constant(K_(a)) of at least about 1×10⁶ M⁻¹ or 10⁷ M⁻¹, or about 10⁸M⁻¹ to 10⁹M⁻¹, or about 10¹° M⁻¹ to 10¹¹ M⁻¹ or higher, and binds to thepredetermined antigen with an affinity that is at least two-fold greaterthan its affinity for binding to a non-specific antigen (e.g., BSA,casein) other than the predetermined antigen or a closely-relatedantigen. The phrases “an antibody recognizing an antigen” and “anantibody specific for an antigen” are used interchangeably herein withthe term “an antibody which binds specifically to an antigen”.

The phrase “specifically bind(s)” or “bind(s) specifically” whenreferring to a peptide refers to a peptide molecule which hasintermediate or high binding affinity, exclusively or predominately, toa target molecule. The phrases “specifically binds to” refers to abinding reaction which is determinative of the presence of a targetprotein in the presence of a heterogeneous population of proteins andother biologics. Thus, under designated assay conditions, the specifiedbinding moieties bind preferentially to a particular target protein anddo not bind in a significant amount to other components present in atest sample. Specific binding to a target protein under such conditionsmay require a binding moiety that is selected for its specificity for aparticular target antigen. A variety of assay formats may be used toselect ligands that are specifically reactive with a particular protein.For example, solid-phase ELISA immunoassays, immunoprecipitation,Biacore and Western blot are used to identify peptides that specificallyreact with CTLA-4. Typically a specific or selective reaction will be atleast twice background signal or noise and more typically more than 10times background.

The term “high affinity” for an IgG antibody refers to an equilibriumassociation constant (K_(a)) of at least about 10⁷M⁻¹, at least about10⁸M⁻¹, at least about 10⁹M⁻¹, at least about 10¹⁰M¹, at least about10¹¹M⁻¹, or at least about 10¹²M⁻¹ or greater, e.g., up to 10¹³M⁻¹ or10¹⁴M⁻¹ or greater. However, “high affinity” binding can vary for otherantibody isotypes.

The term “K_(a)”, as used herein, is intended to refer to theequilibrium association constant of a particular antibody-antigeninteraction. This constant has units of 1/M.

The term “K_(d)”, as used herein, is intended to refer to theequilibrium dissociation constant of a particular antibody-antigeninteraction. This constant has units of M.

The term “k_(a)”, as used herein, is intended to refer to the kineticassociation constant of a particular antibody-antigen interaction. Thisconstant has units of 1/Ms

The term “k_(d)”, as used herein, is intended to refer to the kineticdissociation constant of a particular antibody-antigen interaction. Thisconstant has units of 1/s.

“Particular antibody-antigen interactions” refers to the experimentalconditions under which the equilibrium and kinetic constants aremeasured.

“Isotype” refers to the antibody class (e.g., IgM or IgG1) that isencoded by heavy chain constant region genes.

“Isotype switching” refers to the phenomenon by which the class, orisotype, of an antibody changes from one Ig class to one of the other Igclasses.

“Nonswitched isotype” refers to the isotypic class of heavy chain thatis produced when no isotype switching has taken place; the CH geneencoding the nonswitched isotype is typically the first CH geneimmediately downstream from the functionally rearranged VDJ gene.Isotype switching has been classified as classical or non-classicalisotype switching. Classical isotype switching occurs by recombinationevents which involve at least one switch sequence region in thetransgene. Non-classical isotype switching may occur by, for example,homologous recombination between human σ_(μ) and human Σ_(μ)(δ-associated deletion), Alternative non-classical switching mechanisms,such as intertransgene and/or interchromosomal recombination, amongothers, may occur and effectuate isotype switching.

The term “switch sequence” refers to those DNA sequences responsible forswitch recombination. A “switch donor” sequence, typically a p. switchregion, are 5′ (i.e., upstream) of the construct region to be deletedduring the switch recombination. The “switch acceptor” region arebetween the construct region to be deleted and the replacement constantregion (e.g., γ, ε, etc.). As there is no specific site whererecombination always occurs, the final gene sequence is not typicallypredictable from the construct.

“Glycosylation pattern” is defined as the pattern of carbohydrate unitsthat are covalently attached to a protein, more specifically to animmunoglobulin protein. A glycosylation pattern of a heterologousantibody can be characterized as being substantially similar toglycosylation patterns which occur naturally on antibodies produced bythe species of the non-human transgenic animal, when one of ordinaryskill in the art would recognize the glycosylation pattern of theheterologous antibody as being more similar to said pattern ofglycosylation in the species of the non-human transgenic animal than tothe species from which the CH genes of the transgene were derived.

The term “naturally-occurring” as applied to an object refers to thefact that an object can be found in nature. For example, a polypeptideor polynucleotide sequence that is present in an organism (includingviruses) that can be isolated from a source in nature and which has notbeen intentionally modified by man in the laboratory isnaturally-occurring.

The term “rearranged” refers to a configuration of a heavy chain orlight chain immunoglobulin locus wherein a V segment is positionedimmediately adjacent to a D-J or J segment in a conformation encodingessentially a complete VH or VL domain, respectively. A rearrangedimmunoglobulin gene locus can be identified by comparison to germlineDNA; a rearranged locus has at least one recombined heptamer/nonamerhomology element.

The term “unrearranged” or “germline configuration” in reference to a Vsegment refers to the configuration wherein the V segment is notrecombined so as to be immediately adjacent to a D or J segment.

The term “nucleic acid” is intended to include DNA molecules and RNAmolecules. A nucleic acid can be single-stranded or double-stranded.

The term “isolated nucleic acid” in reference to nucleic acids encodingantibodies or antibody portions (e.g., VH, VL, CDR3) that bind toCTLA-4, is intended to refer to a nucleic acid in which the nucleotidesequences encoding the antibody or antibody portion are free of othernucleotide sequences encoding antibodies or antibody portions that bindantigens other than CTLA-4, which other sequences may naturally flankthe nucleic acid in human genomic DNA. SEQ ID NOs: 4-23 comprise thenucleotide and amino acid sequences comprising the heavy chain (VH) andlight chain (VL) variable regions of the 10D1, 4B6 and 1E2 humananti-CTLA-4 monoclonal antibodies of the invention.

The term “substantially identical,” in the context of two nucleic acidsor polypeptides refers to two or more sequences or subsequences thathave at least about 80%, about 90, about 95% or higher nucleotide oramino acid residue identity, when compared and aligned for maximumcorrespondence, as measured using the following sequence comparisonmethod and/or by visual inspection. For example, the invention providesnucleic acids having sequences that are substantially identical to SEQID NO:1, SEQ ID NO:2. Such “substantially identical” sequences aretypically considered to be homologous. The “substantial identity” canexist over a region of sequence that is at least about 50 residues inlength, over a region of at least about 100 residues, or over a regionat least about 150 residues, or over the full length of the twosequences to be compared. As described below, any two antibody sequencescan only be aligned in one way, by using the numbering scheme in Kabat.Therefore, for antibodies, percent identity has a unique andwell-defined meaning.

Amino acids from the variable regions of the mature heavy and lightchains of immunoglobulins are designated Hx and Lx respectively, where xis a number designating the position of an amino acid according to thescheme of Kabat, Sequences of Proteins of Immunological Interest(National Institutes of Health, Bethesda, Md., 1987 and 1991). Kabatlists many amino acid sequences for antibodies for each subgroup, andlists the most commonly occurring amino acid for each residue positionin that subgroup to generate a consensus sequence. Kabat uses a methodfor assigning a residue number to each amino acid in a listed sequence,and this method for assigning residue numbers has become standard in thefield. Kabat's scheme is extendible to other antibodies not included inhis compendium by aligning the antibody in question with one of theconsensus sequences in Kabat by reference to conserved amino acids. Theuse of the Kabat numbering system readily identifies amino acids atequivalent positions in different antibodies. For example, an amino acidat the L50 position of a human antibody occupies the equivalent positionto an amino acid position L50 of a mouse antibody. Likewise, nucleicacids encoding antibody chains are aligned when the amino acid sequencesencoded by the respective nucleic acids are aligned according to theKabat numbering convention.

The phrase “selectively (or specifically) hybridizes to” refers to thebinding, duplexing, or hybridizing of a molecule to a particularnucleotide sequence under stringent hybridization conditions when thatsequence is present in a complex mixture (e.g., total cellular orlibrary DNA or RNA), wherein the particular nucleotide sequence isdetected at least at about 10 times background. In one embodiment, anucleic acid can be determined to be within the scope of the invention(e.g., is substantially identical to SEQ ID NO:1 or SEQ ID NO:2) by itsability to hybridize under stringent conditions to a nucleic acidotherwise determined to be within the scope of the invention (such asthe exemplary sequences described herein).

The phrase “stringent hybridization conditions” refers to conditionsunder which a probe will hybridize to its target subsequence, typicallyin a complex mixture of nucleic acid, but not to other sequences insignificant amounts (a positive signal (e.g., identification of anucleic acid of the invention) is about 10 times backgroundhybridization). Stringent conditions are sequence-dependent and will bedifferent in different circumstances. Longer sequences hybridizespecifically at higher temperatures. An extensive guide to thehybridization of nucleic acids is found An extensive guide to thehybridization of nucleic acids is found in e.g., Sambrook, ed.,MOLECULAR CLONING: A LABORATORY MANUAL (2ND ED.), Vols. 1-3, Cold SpringHarbor Laboratory, (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,Ausubel, ed. John Wiley & Sons, Inc., New York (1997); LABORATORYTECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY: HYBRIDIZATION WITHNUCLEIC ACID PROBES, Part I. Theory and Nucleic Acid Preparation,Tijssen, ed. Elsevier, N.Y. (1993).

Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength pH. The T_(m) is the temperature (under definedionic strength, pH, and nucleic concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at T, 50% ofthe probes are occupied at equilibrium). Stringent conditions will bethose in which the salt concentration is less than about 1.0 M sodiumion, typically about 0.01 to 1.0 M sodium ion concentration (or othersalts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. forshort probes (e.g., 10 to 50 nucleotides) and at least about 60° C. forlong probes (e.g., greater than 50 nucleotides). Stringent conditionsmay also be achieved with the addition of destabilizing agents such asformamide as described in Sambrook (cited below). For high stringencyhybridization, a positive signal is at least two times background,preferably 10 times background hybridization. Exemplary high stringencyor stringent hybridization conditions include: 50% formamide, 5×SSC and1% SDS incubated at 42° C. or 5×SSC and 1% SDS incubated at 65° C., witha wash in 0.2×SSC and 0.1% SDS at 65° C. For selective or specifichybridization, a positive signal (e.g., identification of a nucleic acidof the invention) is about 10 times background hybridization. Stringenthybridization conditions that are used to identify nucleic acids withinthe scope of the invention include, e.g., hybridization in a buffercomprising 50% formamide, 5×SSC, and 1% SDS at 42° C., or hybridizationin a buffer comprising 5×SSC and 1% SDS at 65° C., both with a wash of0.2×SSC and 0.1% SDS at 65° C. In the present invention, genomic DNA orcDNA comprising nucleic acids of the invention can be identified instandard Southern blots under stringent conditions using the nucleicacid sequences disclosed here. Additional stringent conditions for suchhybridizations (to identify nucleic acids within the scope of theinvention) are those which include a hybridization in a buffer of 40%formamide, 1 M NaCl, 1% SDS at 37° C.

However, the selection of a hybridization format is not critical—it isthe stringency of the wash conditions that set forth the conditionswhich determine whether a nucleic acid is within the scope of theinvention. Wash conditions used to identify nucleic acids within thescope of the invention include, e.g.: a salt concentration of about 0.02molar at pH 7 and a temperature of at least about 50° C. or about 55° C.to about 60° C.; or, a salt concentration of about 0.15 M NaCl at 72° C.for about 15 minutes; or, a salt concentration of about 0.2×SSC at atemperature of at least about 50° C. or about 55° C. to about 60° C. forabout 15 to about 20 minutes; or, the hybridization complex is washedtwice with a solution with a salt concentration of about 2×SSCcontaining 0.1% SDS at room temperature for 15 minutes and then washedtwice by 0.1×SSC containing 0.1% SDS at 68° C. for 15 minutes; or,equivalent conditions. See Sambrook, Tijssen and Ausubel for adescription of SSC buffer and equivalent conditions.

The nucleic acids of the invention be present in whole cells, in a celllysate, or in a partially purified or substantially pure form. A nucleicacid is “isolated” or “rendered substantially pure” when purified awayfrom other cellular components or other contaminants, e.g., othercellular nucleic acids or proteins, by standard techniques, includingalkaline/SDS treatment, CsCl banding, column chromatography, agarose gelelectrophoresis and others well known in the art. see, e.g., Sambrook,Tijssen and Ausubel. The nucleic acid sequences of the invention andother nucleic acids used to practice this invention, whether RNA, cDNA,genomic DNA, or hybrids thereof, may be isolated from a variety ofsources, genetically engineered, amplified, and/or expressedrecombinantly. Any recombinant expression system can be used, including,in addition to bacterial, e.g., yeast, insect or mammalian systems.Alternatively, these nucleic acids can be chemically synthesized invitro. Techniques for the manipulation of nucleic acids, such as, e.g.,subcloning into expression vectors, labeling probes, sequencing, andhybridization are well described in the scientific and patentliterature, see, e.g., Sambrook, Tijssen and Ausubel. Nucleic acids canbe analyzed and quantified by any of a number of general means wellknown to those of skill in the art. These include, e.g., analyticalbiochemical methods such as NMR, spectrophotometry, radiography,electrophoresis, capillary electrophoresis, high performance liquidchromatography (HPLC), thin layer chromatography (TLC), andhyperdiffusion chromatography, various immunological methods, such asfluid or gel precipitin reactions, immunodiffusion (single or double),immunoelectrophoresis, radioimmunoassays (RIAs), enzyme-linkedimmunosorbent assays (ELISAs), immuno-fluorescent assays, Southernanalysis, Northern analysis, dot-blot analysis, gel electrophoresis(e.g., SDS-PAGE), RT-PCR, quantitative PCR, other nucleic acid or targetor signal amplification methods, radiolabeling, scintillation counting,and affinity chromatography.

The nucleic acid compositions of the present invention, while often in anative sequence (except for modified restriction sites and the like),from either cDNA, genomic or mixtures may be mutated, thereof inaccordance with standard techniques to provide gene sequences. Forcoding sequences, these mutations, may affect amino acid sequence asdesired. In particular, DNA sequences substantially homologous to orderived from native V, D, J, constant, switches and other such sequencesdescribed herein are contemplated (where “derived” indicates that asequence is identical or modified from another sequence).

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For instance, apromoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the sequence. With respect to transcriptionregulatory sequences, operably linked means that the DNA sequences beinglinked are contiguous and, where necessary to join two protein codingregions, contiguous and in reading frame. For switch sequences, operablylinked indicates that the sequences are capable of effecting switchrecombination.

The term “vector” is intended to refer to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of vector is a “plasmid”, which refers to a circulardouble stranded DNA loop into which additional DNA segments may beligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The term “recombinant host cell” (or simply “host cell”) refers to acell into which a recombinant expression vector has been introduced. Itshould be understood that such terms are intended to refer not only tothe particular subject cell but to the progeny of such a cell. Becausecertain modifications may occur in succeeding generations due to eithermutation or environmental influences, such progeny may not, in fact, beidentical to the parent cell, but are still included within the scope ofthe term “host cell” as used herein.

The term “minilocus transgene” refers to a transgene that comprises aportion of the genomic immunoglobulin locus having at least one internal(i.e., not at a terminus of the portion) deletion of a non-essential DNAportion (e.g., intervening sequence; intron or portion thereof) ascompared to the naturally-occurring germline Ig locus.

A “label” is a composition detectable by spectroscopic, photochemical,biochemical, immunochemical, or chemical means. For example, usefullabels include ³²P, fluorescent dyes, electron-dense reagents, enzymes(e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptensand proteins for which antisera or monoclonal antibodies are available(e.g., the polypeptides of the invention can be made detectable, e.g.,by incorporating a radiolabel into the peptide, and used to detectantibodies specifically reactive with the peptide).

The term “sorting” in the context of cells as used herein to refers toboth physical sorting of the cells, as can be accomplished using, e.g.,a fluorescence activated cell sorter, as well as to analysis of cellsbased on expression of cell surface markers, e.g., FACS analysis in theabsence of sorting.

The phrase “immune cell response” refers to the response of immunesystem cells to external or internal stimuli (e.g., antigen, cytokines,chemokines, and other cells) producing biochemical changes in the immunecells that result in immune cell migration, killing of target cells,phagocytosis, production of antibodies, other soluble effectors of theimmune response, and the like.

The terms “T lymphocyte response” and “T lymphocyte activity” are usedhere interchangeably to refer to the component of immune responsedependent on T lymphocytes (i.e., the proliferation and/ordifferentiation of T lymphocytes into helper, cytotoxic killer, orsuppressor T lymphocytes, the provision of signals by helper Tlymphocytes to B lymphocytes that cause or prevent antibody production,the killing of specific target cells by cytotoxic T lymphocytes, and therelease of soluble factors such as cytokines that modulate the functionof other immune cells).

The term “immune response” refers to the concerted action oflymphocytes, antigen presenting cells, phagocytic cells, granulocytes,and soluble macromolecules produced by the above cells or the liver(including antibodies, cytokines, and complement) that results inselective damage to, destruction of, or elimination from the human bodyof invading pathogens, cells or tissues infected with pathogens,cancerous cells, or, in cases of autoimmunity or pathologicalinflammation, normal human cells or tissues.

Components of an immune response may be detected in vitro by variousmethods that are well known to those of ordinary skill in the art. Forexample, (1) cytotoxic T lymphocytes can be incubated with radioactivelylabeled target cells and the lysis of these target cells detected by therelease of radioactivity, (2) helper T lymphocytes can be incubated withantigens and antigen presenting cells and the synthesis and secretion ofcytokines measured by standard methods (Windhagen A; et al., 1995,Immunity 2(4): 373-80), (3) antigen presenting cells can be incubatedwith whole protein antigen and the presentation of that antigen on MHCdetected by either T lymphocyte activation assays or biophysical methods(Harding et al., 1989, Proc. Natl. Acad. Sci., 86: 4230-4), (4) mastcells can be incubated with reagents that cross-link their Fc-epsilonreceptors and histamine release measured by enzyme immunoassay(Siraganian, et al., 1983, TIPS 4: 432-437).

Similarly, products of an immune response in either a model organism(e.g., mouse) or a human patient can also be detected by various methodsthat are well known to those of ordinary skill in the art. For example,(1) the production of antibodies in response to vaccination can bereadily detected by standard methods currently used in clinicallaboratories, e.g., an ELISA; (2) the migration of immune cells to sitesof inflammation can be detected by scratching the surface of skin andplacing a sterile container to capture the migrating cells over scratchsite (Peters et al., 1988, Blood 72: 1310-5); (3) the proliferation ofperipheral blood mononuclear cells in response to mitogens or mixedlymphocyte reaction can be measured using ³H-thymidine; (4) thephagocitic capacity of granulocytes, macrophages, and other phagocytesin PBMCs can be measured by placing PMBCs in wells together with labeledparticles (Peters et al., 1988); and (5) the differentation of immunesystem cells can be measured by labeling PBMCs with antibodies to CDmolecules such as CD4 and CD8 and measuring the fraction of the PBMCsexpressing these markers.

As used herein, the phrase “signal transduction pathway” or “signaltransduction event” refers to at least one biochemical reaction, butmore commonly a series of biochemical reactions, which result frominteraction of a cell with a stimulatory compound or agent. Thus, theinteraction of a stimulatory compound with a cell generates a “signal”that is transmitted through the signal transduction pathway, ultimatelyresulting in a cellular response, e.g., an immune response describedabove.

A signal transduction pathway refers to the biochemical relationshipbetween a variety of signal transduction molecules that play a role inthe transmission of a signal from one portion of a cell to anotherportion of a cell. Signal transduction molecules of the presentinvention include, for example, MAb 147.1 of the invention. As usedherein, the phrase “cell surface receptor” includes molecules andcomplexes of molecules capable of receiving a signal and thetransmission of such a signal across the plasma membrane of a cell. Anexample of a “cell surface receptor” of the present invention is the Tcell receptor (TCR) or the B7 ligands of CTLA-4.

A signal transduction pathway in a cell can be initiated by interactionof a cell with a stimulator that is inside or outside of the cell. If anexterior (i.e., outside of the cell) stimulator (e.g., an MHC-antigencomplex on an antigen presenting cell) interacts with a cell surfacereceptor (e.g., a T cell receptor), a signal transduction pathway cantransmit a signal across the cell's membrane, through the cytoplasm ofthe cell, and in some instances into the nucleus. If an interior (e.g.,inside the cell) stimulator interacts with an intracellular signaltransduction molecule, a signal transduction pathway can result intransmission of a signal through the cell's cytoplasm, and in someinstances into the cell's nucleus.

Signal transduction can occur through, e.g., the phosphorylation of amolecule; non-covalent allosteric interactions; complexing of molecules;the conformational change of a molecule; calcium release; inositolphosphate production; proteolytic cleavage; cyclic nucleotide productionand diacylglyceride production. Typically, signal transduction occursthrough phosphorylating a signal transduction molecule.

The term “nonspecific T cell activation” refers to the stimulation of Tcells independent of their antigenic specificity.

Production of Human Antibodies to CTLA-4

The monoclonal antibodies (mAbs) and the human sequence antibodies ofthe invention can be produced by a variety of techniques, includingconventional monoclonal antibody methodology e.g., the standard somaticcell hybridization technique of Kohler and Milstein, Nature 256: 495(1975). Any technique for producing monoclonal antibody can be employede.g., viral or oncogenic transformation of B lymphocytes. One animalsystem for preparing hybridomas is the murine system. Hybridomaproduction in the mouse is a very well-established procedure.Immunization protocols and techniques for isolation of immunizedsplenocytes for fusion are known in the art. Fusion partners (e.g.,murine myeloma cells) and fusion procedures are also known (see, e.g.,Harlow and Lane (1988), Antibodies, A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor N.Y.).

Human monoclonal antibodies and human sequence antibodies directedagainst human CTLA-4 can be generated using transgenic mice carrying ahuman immune system rather than the mouse system. These transgenic mice,also referred to herein as “HuMAb-Mouse™”, contain a humanimmunoglobulin gene miniloci that encodes unrearranged human heavy (μand γ) and κ light chain immunoglobulin sequences, together withtargeted mutations that inactivate the endogenous μ and κ chain loci(Lonberg, N. et al. (1994) Nature 368(6474): 856-859 and U.S. Pat. No.5,770,429). Accordingly, the mice exhibit reduced expression of mouseIgM or K, and in response to immunization, the introduced human heavyand light chain transgenes undergo class switching and somatic mutationto generate high affinity human IgGκ monoclonal (Lonberg, N. et al.(1994), supra; reviewed in Lonberg, N. (1994) Handbook of ExperimentalPharmacology 113:49-101; Lonberg, N. and Huszar, D. (1995) Intern. Rev.Immunol. Vol. 13: 65-93, and Harding, F. and Lonberg, N. (1995) Ann.N.Y. Acad. Sci. 764:536-546). The preparation of transgenic mice isdescribed in detail Section II below and in Taylor, L. et al., (1992)Nucleic Acids Research 20:6287-6295; Chen, J. et al. (1993)International Immunology 5: 647-656; Tuaillon et al. (1993) Proc. Natl.Acad. Sci. USA 90:3720-3724; Choi et al. (1993) Nature Genetics4:117-123; Chen, J. et al. (1993) EMBO J. 12: 821-830; Tuaillon et al.,(1994) J. Immunol. 152:2912-2920; Lonberg et al., (1994) Nature368(6474): 856-859; Lonberg, N. (1994) Handbook of ExperimentalPharmacology 113:49-101; Taylor, L. et al., (1994) InternationalImmunology 6: 579-591; Lonberg, N. and Huszar, D. (1995) Intern. Rev.Immunol, Vol. 13: 65-93; Harding, F. and Lonberg, N. (1995) Ann. N.Y.Acad. Sci. 764:536-546; Fishwild, D. et al. (1996) Nature Biotechnology14: 845-851. See further, U.S. Pat. Nos. 5,625,126 and 5,770,429, bothto Lonberg and Kay, and GenPharm International; U.S. Pat. No. 5,545,807to Surani et al.; International Publication Nos. WO 98/24884, publishedon Jun. 11, 1998; WO 94/25585, published Nov. 10, 1994; WO 93/1227,published Jun. 24, 1993; WO 92/22645, published Dec. 23, 1992; WO92/03918, published Mar. 19, 1992. Alternatively, the CMD and HCo12transgenes, described in Examples 1 and 2, below, can be used togenerate human anti-CTLA-4 antibodies.

Detailed procedures to generate fully human monoclonal antibodies toCTLA-4 are described in the Examples below. Cumulative experience withvarious antigens has shown that the transgenic mice respond wheninitially immunized intraperitoneally (IP) with antigen in completeFreund's adjuvant, followed by every other week IP immunizations (up toa total of 6) with antigen in incomplete Freund's adjuvant. However,adjuvants other than Freund's are also found to be effective. Inaddition, whole cells in the absence of adjuvant are found to be highlyimmunogenic. The immune response can be monitored over the course of theimmunization protocol with plasma samples being obtained by retroorbitalbleeds. The plasma can be screened by ELISA (as described below), andmice with sufficient titers of anti-CTLA-4 human immunoglobulin can beused for fusions. Mice can be boosted intravenously with antigen 3 daysbefore sacrifice and removal of the spleen. It is expected that 2-3fusions for each immunization may need to be performed. Between 6 and 24mice are typically immunized for each antigen. Usually both HCo7 andHCo12 strains are used. In addition, both HCo7 and HCo12 transgene canbe bred together into a single mouse having two different human heavychain transgenes.

To purify human anti-CTLA-4 antibodies, selected hybridomas can be grownin two-liter spinner-flasks for monoclonal antibody purification.Supernatants can be filtered and concentrated before affinitychromatography with protein A-sepharose (Pharmacia, Piscataway, N.J.).Fluted IgG can be checked by gel electrophoresis and high performanceliquid chromatography to ensure purity. The buffer solution can beexchanged into PBS, and the concentration can be determined by OD280using 1.43 extinction coefficient. The monoclonal antibodies can bealiquoted and stored at −80° C.

To determine if the selected human anti-CTLA-4 monoclonal antibodiesbind to unique epitopes, each antibody can be biotinylated usingcommercially available reagents (Pierce, Rockford, Ill.). Competitionstudies using unlabeled monoclonal antibodies and biotinylatedmonoclonal antibodies can be performed using CTLA-4 coated-ELISA platesas described above. Biotinylated MAb binding can be detected with astrep-avidin-alkaline phosphatase probe.

To determine the isotype of purified antibodies, isotype ELISAs can beperformed. Wells of microtiter plates can be coated with 1 μg/ml ofanti-human IgG overnight at 4° C. After blocking with 1% BSA, the platesare reacted with 1 μg/ml or less of monoclonal antibodies or purifiedisotype controls, at ambient temperature for one to two hours. The wellscan then be reacted with either human IgG1 or human IgM-specificalkaline phosphatase-conjugated probes. Plates are developed andanalyzed as described above.

To demonstrate binding of monoclonal antibodies to live cells expressingthe CTLA-4, flow cytometry can be used. Briefly, cell lines expressingCTLA-4 (grown under standard growth conditions) are mixed with variousconcentrations of monoclonal antibodies in PBS containing 0.1% BSA and10% fetal calf serum, and incubated at 37° C. for 1 hour. After washing,the cells are reacted with Fluorescein-labeled anti-human IgG antibodyunder the same conditions as the primary antibody staining. The samplescan be analyzed by FACScan instrument using light and side scatterproperties to gate on single cells. An alternative assay usingfluorescence microscopy may be used (in addition to or instead of) theflow cytometry assay. Cells can be stained exactly as described aboveand examined by fluorescence microscopy. This method allowsvisualization of individual cells, but may have diminished sensitivitydepending on the density of the antigen.

Anti-CTLA-4 human IgGs can be further tested for reactivity with CTLA-4antigen by Western blotting. Briefly, cell extracts from cellsexpressing CTLA-4 can be prepared and subjected to sodium dodecylsulfate polyacrylamide gel electrophoresis. After electrophoresis, theseparated antigens are transferred to nitrocellulose membranes, blockedwith 10% fetal calf serum, and probed with the monoclonal antibodies tobe tested. Human IgG binding can be detected using anti-human IgGalkaline phosphatase and developed with BCIP/NBT substrate tablets(Sigma Chem. Co., St. Louis, Mo.).

Production of Transgenic Non-Human Animals that Generate HumanMonoclonal Anti-CTLA-4 Antibodies

The present invention also provides transgenic non-human animals, e.g.,a transgenic mice, which are capable of expressing human monoclonalantibodies that specifically bind to CTLA-4. High affinity humansequence antibodies are also provided. Some transgenic non-humananimals, e.g., the transgenic mice, have a genome comprising a humanheavy chain transgene and a light chain transgene. Some transgenicnon-human animals are immunized with a purified or enriched preparationof CTLA-4 antigen and/or cells expressing CTLA-4. Some transgenicnon-human animals are capable of producing multiple isotypes of humanmonoclonal antibodies to CTLA-4 (e.g., IgG, IgA and/or IgE) byundergoing V-D-J recombination and isotype switching. Isotype switchingmay occur by, e.g., classical or non-classical isotype switching.

The design of a transgenic non-human animal that responds to foreignantigen stimulation with a heterologous antibody repertoire, requiresthat the heterologous immunoglobulin transgenes contained within thetransgenic animal function correctly throughout the pathway of B-celldevelopment. In some mice, correct function of a heterologous heavychain transgene includes isotype switching. Accordingly, the transgenesof the invention are constructed so as to produce isotype switching andone or more of the following: (1) high level and cell-type specificexpression, (2) functional gene rearrangement, (3) activation of andresponse to allelic exclusion, (4) expression of a sufficient primaryrepertoire, (5) signal transduction, (6) somatic hypermutation, and (7)domination of the transgene antibody locus during the immune response.

Not all of the foregoing criteria need be met. For example, intransgenic animal in which the endogenous immunoglobulin loci of thetransgenic animals are functionally disrupted, the transgene need notactivate allelic exclusion. Further, in transgenic animals in which thetransgene comprises a functionally rearranged heavy and/or light chainimmunoglobulin gene, the second criteria of functional generearrangement is unnecessary, at least for that transgene which isalready rearranged. For background on molecular immunology, See, e.g.,Fundamental Immunology, 4th edition (1998), Paul, William E., ed.Lippencott-Raven Press, N.Y.

Some transgenic non-human animals used to generate the human monoclonalantibodies of the invention contain rearranged, unrearranged or acombination of rearranged and unrearranged heterologous immunoglobulinheavy and light chain transgenes in the germline of the transgenicanimal. Each of the heavy chain transgenes comprises at least one CHgene. In addition, the heavy chain transgene can contain functionalisotype switch sequences, which are capable of supporting isotypeswitching of a heterologous transgene encoding multiple CH genes in theB-cells of the transgenic animal. Such switch sequences can be thosewhich occur naturally in the germline immunoglobulin locus from thespecies that serves as the source of the transgene CH genes, or suchswitch sequences can be derived from those which occur in the speciesthat is to receive the transgene construct (the transgenic animal). Forexample, a human transgene construct that is used to produce atransgenic mouse may produce a higher frequency of isotype switchingevents if it incorporates switch sequences similar to those that occurnaturally in the mouse heavy chain locus, as presumably the mouse switchsequences are optimized to function with the mouse switch recombinaseenzyme system, whereas the human switch sequences are not. Switchsequences can be isolated and cloned by conventional cloning methods, orcan be synthesized de novo from overlapping synthetic oligonucleotidesdesigned on the basis of published sequence information relating toimmunoglobulin switch region sequences (Mills et al., Nucl. Acids Res.15:7305-7316 (1991); Sideras et al., Intl. Immunol. 1:631-642 (1989).

For each of the foregoing transgenic animals, functionally rearrangedheterologous heavy and light chain immunoglobulin transgenes are foundin a significant fraction of the B-cells of the transgenic animal (atleast 10 percent).

The transgenes used to generate the transgenic animals of the inventioninclude a heavy chain transgene comprising DNA encoding at least onevariable gene segment, one diversity gene segment, one joining genesegment and at least one constant region gene segment. Theimmunoglobulin light chain transgene comprises DNA encoding at least onevariable gene segment, one joining gene segment and at least oneconstant region gene segment. The gene segments encoding the light andheavy chain gene segments are heterologous to the transgenic non-humananimal in that they are derived from, or correspond to, DNA encodingimmunoglobulin heavy and light chain gene segments from a species notconsisting of the transgenic non-human animal. In one aspect of theinvention, the transgene is constructed such that the individual genesegments are unrearranged, i.e., not rearranged so as to encode afunctional immunoglobulin light or heavy chain. Such unrearrangedtransgenes support recombination of the V, D, and J gene segments(functional rearrangement) and preferably support incorporation of allor a portion of a D region gene segment in the resultant rearrangedimmunoglobulin heavy chain within the transgenic non-human animal whenexposed to CTLA-4 antigen.

Such transgenes typically comprise a substantial portion of the C, D,and J segments as well as a subset of the V gene segments. In suchtransgene constructs, the various regulatory sequences, e.g. promoters,enhancers, class switch regions, splice-donor and splice-acceptorsequences for RNA processing, recombination signals and the like,comprise corresponding sequences derived from the heterologous DNA. Suchregulatory sequences may be incorporated into the transgene from thesame or a related species of the non-human animal used in the invention.For example, human immunoglobulin gene segments may be combined in atransgene with a rodent immunoglobulin enhancer sequence for use in atransgenic mouse. Alternatively, synthetic regulatory sequences may beincorporated into the transgene, wherein such synthetic regulatorysequences are not homologous to a functional DNA sequence that is knownto occur naturally in the genomes of mammals. Synthetic regulatorysequences are designed according to consensus rules, such as, forexample, those specifying the permissible sequences of a splice-acceptorsite or a promoter/enhancer motif. The transgene may comprise aminilocus.

Some transgenic animals used to generate human antibodies to CTLA-4contain at least one, typically 2-10, and sometimes 25-50 or more copiesof the transgene described in Example 37 of U.S. Pat. No. 5,770,429, orthe transgene described in Example 2 below (e.g., HCo12), at least onecopy of a light chain transgene described in Examples 38 of U.S. Pat.No. 5,770,429, two copies of the Cmu deletion described in Example 1below, and two copies of the Jkappa deletion described in Example 9 ofU.S. Pat. No. 5,770,429. The resultant animals are injected withantigens and used for production of human monoclonal antibodies againstthese antigens.

Some transgenic animals exhibit immunoglobulin production with asignificant repertoire, ideally substantially similar to that of anative mouse. Thus, for example, animals in which the endogenous Iggenes have been inactivated, the total immunoglobulin levels range fromabout 0.1 to about 10 mg/ml of serum.

The immunoglobulins expressed by the transgenic mice typically recognizeabout one-half or more of highly antigenic proteins, e.g.,staphylococcus protein A. Typically, the immunoglobulins exhibit anassociation constant for preselected antigens of at least about 10⁷M⁻¹,10⁸M⁻¹, 10⁹M⁻¹, 10¹⁰M⁻¹, 10¹¹M⁻¹, 10¹²M⁻¹, 10¹³M⁻¹, or greater.

The transgenic mice of the present invention can be immunized with apurified or enriched preparation of human CTLA-4 antigen (or antigenicfragment thereof) and/or cells expressing human CTLA-4 as describedpreviously. The mice produce B cells that undergo class-switching viaintratransgene switch recombination (cis-switching) and expressimmunoglobulins reactive with CTLA-4. The immunoglobulins can be humansequence antibodies, wherein the heavy and light chain polypeptides areencoded by human transgene sequences, which may include sequencesderived by somatic mutation and V region recombinatorial joints, as wellas germline-encoded sequences; these human sequence immunoglobulins canbe referred to as being substantially identical to a polypeptidesequence encoded by a human VL or VH gene segment and a human JL or JHsegment, even though other non-germline sequences may be present as aresult of somatic mutation and differential V-J and V-D-J recombinationjoints. With respect to such human sequence antibodies, the variableregions of each chain are typically at least 80 percent encoded by humangermline V, J, and, in the case of heavy chains, D, gene segments;frequently at least 85 percent of the variable regions are encoded byhuman germline sequences present on the transgene; often 90 or 95percent or more of the variable region sequences are encoded by humangermline sequences present on the transgene. However, since non-germlinesequences are introduced by somatic mutation and VJ and VDJ joining, thehuman sequence antibodies frequently have some variable region sequences(and less frequently constant region sequences) which are not encoded byhuman V, D, or J gene segments as found in the human transgene(s) in thegermline of the mice. Typically, such non-germline sequences (orindividual nucleotide positions) cluster in or near CDRs, or in regionswhere somatic mutations are known to cluster.

The human sequence antibodies which bind to the predetermined antigencan result from isotype switching, such that human antibodies comprisinga human sequence γ chain (such as γ1, γ2, γ3, or γ4) and a humansequence light chain (such as kappa or lambda) are produced. Suchisotype-switched human sequence antibodies often contain one or moresomatic mutation(s), typically in the variable region and often in orwithin about 10 residues of a CDR) as a result of affinity maturationand selection of B cells by antigen, particularly subsequent tosecondary (or subsequent) antigen challenge. Some high affinity humansequence antibodies have equilibrium association constants of at leastabout 1×10⁷ M⁻¹, or at least about 1×10⁸ M⁻¹, or more than about 1×10⁹M⁻¹, or 5×10⁹ M⁻¹ to 1×10¹¹ M⁻¹ or greater.

Another aspect of the invention pertains to the B cells from such micewhich can be used to generate hybridomas expressing human monoclonalantibodies which bind with high affinity (e.g., having associationconstant of greater than 10⁷M⁻¹) to CTLA-4. These hybridomas are used togenerate a composition comprising an immunoglobulin having anassociation constant (Ka) of at least 10⁷ M⁻¹ for binding CTLA-4. Suchimmunoglobulin contains a human sequence light chain composed of a lightchain variable region having a polypeptide sequence which issubstantially identical to a polypeptide sequence encoded by a human Vkor Vλ gene segment and a human Jk or Jλ segment, and a light chainconstant region having a polypeptide sequence which is substantiallyidentical to a polypeptide sequence encoded by a human Ck or Cλ genesegment. It also contains a human sequence heavy chain composed of aheavy chain variable region having a polypeptide sequence which issubstantially identical to a polypeptide sequence encoded by a human VHgene segment, optionally a D region, and a human JH segment, and aconstant region having a polypeptide sequence which is substantiallyidentical to a polypeptide sequence encoded by a human CH gene segment.

The invention also provides human monoclonal antibodies and humansequence antibodies to human CTLA-4 derivatized or linked to anotherfunctional molecule, e.g., another peptide or protein (e.g., a cytokine,a cytotoxic agent, an immune stimulatory or inhibitory agent, a Fab′fragment, and the like, as discussed above) to generate a bispecific ormultispecific molecule which binds to multiple binding sites or targetepitopes. For example, an antibody or antigen-binding portion of theinvention can be functionally linked (e.g., by chemical coupling,genetic fusion, noncovalent association or otherwise) to one or moreother binding molecules, such as another antibody, antibody fragment,peptide or binding mimetic.

Accordingly, the present invention includes bispecific and multispecificcomposition comprising at least one human sequence antibody or antigenbinding fragment with a first binding specificity for human CTLA-4 and asecond binding specificity for a second target epitope. The secondtarget epitope can be an Fe receptor, e.g., human FcγRI or a human Fcγreceptor. Therefore, the invention includes bispecific and multispecificmolecules capable of binding both to FcγR1, FcγR or FcεR. expressingeffector cells (e.g., monocytes, macrophages or polymorphonuclear cells(PMNs)), and to target cells expressing human CTLA-4. Thesemultispecific (e.g., bispecific or multispecific) molecules target humanCTLA-4 expressing cells to effector cells, and trigger Fcreceptor-mediated effector cell activities, such as phagocytosis of ahuman CTLA-4-expressing cells, antibody dependent cell-mediatedcytotoxicity (ADCC), cytokine release, or generation of superoxideanion.

The bispecific and multispecific molecules of the invention can comprisea binding specificity at least one antibody, or an antibody fragmentthereof, including, e.g., an Fab, Fab′, F(ab′)₂, Fv, or a single chainFv. The antibody may also be a light chain or heavy chain dimer, or anyminimal fragment thereof such as a Fv or a single chain construct asdescribed in, e.g., Ladner et al. U.S. Pat. No. 4,946,778. Bispecificand multispecific molecules of the invention can comprise a bindingspecificity for an FcγR or an FcγR present on the surface of an effectorcell, and a second binding specificity for a target cell antigen, e.g.,human CTLA-4.

The binding specificity for an Fc receptor is provided by a monoclonalantibody, the binding of which is not blocked by human immunoglobulin G(IgG). As used herein, the term “IgG receptor” refers to any of theeight γ-chain genes located on chromosome 1. These genes encode a totalof twelve transmembrane or soluble receptor isoforms which are groupedinto three Fcγ receptor classes: FcγRI (CD64), FcγRII (CD32), andFcγRIII (CD16). For example, the Fcγ receptor can be a human highaffinity FcγRI. The human FcγRI is a 72 kDa molecule, which shows highaffinity for monomeric IgG (10⁸ to 10⁹ M⁻¹).

The production and characterization of these preferred monoclonalantibodies are described by Fanger et al. in PCT application WO 88/00052and in U.S. Pat. No. 4,954,617. These antibodies bind to an epitope ofFcγRI, FcγRII or FcγRIII at a site which is distinct from the Fcγbinding site of the receptor and, thus, their binding is not blockedsubstantially by physiological levels of IgG. Specific anti-FcγRIantibodies useful in this invention are MAb 22, MAb 32, MAb 44, MAb 62and MAb 197. The hybridoma producing MAb 32 is available from theAmerican Type Culture Collection, ATCC Accession No. HB9469. Anti-FcγRIMAb 22, F(ab′)₂ fragments of MAb 22, and can be obtained from Medarex,Inc. (Annandale, N.J.). In other embodiments, the anti-Fey receptorantibody is a humanized form of monoclonal antibody 22 (H22). Theproduction and characterization of the H22 antibody is described inGraziano (1995) J. Immunol. 155:4996-5002 and PCT/US93/10384, The H22antibody producing cell line was deposited at the American Type CultureCollection on Nov. 4, 1992 under the designation HA022CL1 and has theaccession no. CRL 11177.

The binding specificity for an Fc receptor can also be provided by anantibody that binds to a human IgA receptor, e.g., an Fc-alpha receptor(FcαR (CD89)). Preferably, the antibody binds to a human IgA receptor ata site that is not blocked by endogenous IgA. The term “IgA receptor” isintended to include the gene product of one α-gene (FcαRI) located onchromosome 19. This gene is known to encode several alternativelyspliced transmembrane isoforms of 55 to 110 kDa. FcαRI (CD89) isconstitutively expressed on monocytes/macrophages, eosinophilic andneutrophilic granulocytes, but not on non-effector cell populations.FcαRI has medium affinity (≈5×10⁷ M⁻¹) for both IgA1 and IgA2, which isincreased upon exposure to cytokines such as G-CSF or GM-CSF (Morton(1996) Critical Reviews in Immunology 16:423-440). Four FcαRI-specificmonoclonal antibodies, identified as A3, A59, A62 and A77, which bindFcαRI outside the IgA ligand binding domain, have been described by,e.g., Monteiro (1992) J. Immunol. 148:1764.

Bispecific and multispecific molecules of the invention can furthercomprise a binding specificity which recognizes, e.g., binds to, atarget cell antigen, e.g. human CTLA-4. The binding specificity isprovided by a human sequence antibody or a human monoclonal antibody ofthe present invention.

An “effector cell specific antibody” as used herein refers to anantibody or functional antibody fragment that binds the Fe receptor ofeffector cells. Preferred antibodies for use in the subject inventionbind the Fe receptor of effector cells at a site which is not bound byendogenous immunoglobulin.

As used herein, the term “effector cell” refers to an immune cell whichis involved in the effector phase of an immune response, as opposed tothe cognitive and activation phases of an immune response. Exemplaryimmune cells include a cell of a myeloid or lymphoid origin, e.g.,lymphocytes (e.g., B cells and T cells including cytolytic T cells(CTLs)), killer cells, natural killer cells, macrophages, monocytes,eosinophils, neutrophils, polymorphonuclear cells, granulocytes, mastcells, and basophils. Effector cells express specific Fc receptors andcarry out specific immune functions. An effector cell can induceantibody-dependent cell-mediated cytotoxicity (ADCC), e.g., a neutrophilcapable of inducing ADCC. For example, monocytes, macrophages,neutrophils, eosinophils, and lymphocytes which express FcαR areinvolved in specific killing of target cells and presenting antigens toother components of the immune system, or binding to cells that presentantigens. An effector cell can also phagocytose a target antigen, targetcell, or microorganism.

The expression of a particular FcR on an effector cell can be regulatedby humoral factors such as cytokines. For example, expression of FcγRIhas been found to be up-regulated by interferon gamma (IFN-γ). Thisenhanced expression increases cytotoxic activity (including, e.g.,phagocytosis) by FcγRI-bearing cells against target cells.

“Target cell” shall mean any undesirable cell in a subject (e.g., ahuman or animal) that can be targeted by a composition (e.g., a humansequence antibody or a human monoclonal antibody of the invention, abispecific or a multispecific molecule of the invention). The targetcell can be a cell expressing or overexpressing human CTLA-4. Cellsexpressing human CTLA-4 can include tumor cells, e.g. lymphomas.

In addition to human sequence antibodies and human monoclonal antibodiesof the invention, other antibodies can be also be employed in thebispecific or multispecific molecules of the invention, including, e.g.,murine, chimeric and humanized monoclonal antibodies.

Chimeric mouse-human monoclonal antibodies (i.e., chimeric antibodies)can be produced by recombinant DNA techniques known in the art. Forexample, a gene encoding the Fc constant region of a murine (or otherspecies) monoclonal antibody molecule is digested with restrictionenzymes to remove the region encoding the murine Fc, and the equivalentportion of a gene encoding a human Fc constant region is substituted.(See, e.g., Robinson et al., International Patent PublicationPCT/US86/02269; Akira, et al., European Patent Application 184,187;Taniguchi, M., European Patent Application 171,496; Morrison et al.,European Patent Application 173,494; Neuberger et al., InternationalApplication WO 86/01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabillyet al., European Patent Application 125,023; Better (1988) Science240:1041-1043; Liu (1987) PNAS 84:3439-3443; Liu (1987) J. Immunol.139:3521-3526; Sun (1987) PNAS 84:214-218; Nishimura (1987) Cane. Res.47:999-1005; Wood (1985) Nature 314:446-449; Shaw (1988) J. Natl. CancerInst. 80:1553-1559).

The chimeric antibody can be further humanized by replacing sequences ofthe Fv variable region which are not directly involved in antigenbinding with equivalent sequences from human Fv variable regions.General reviews of humanized chimeric antibodies are provided byMorrison (1985) Science 229:1202-1207 and by Oi (1986) BioTechniques4:214. Those methods include isolating, manipulating, and expressing thenucleic acid sequences that encode all or part of immunoglobulin Fvvariable regions from at least one of a heavy or light chain. Sources ofsuch nucleic acid are well known to those skilled in the art and, forexample, may be obtained from 7E3, an anti-GPII_(b)III_(a) antibodyproducing hybridoma. The recombinant DNA encoding the chimeric antibody,or fragment thereof, can then be cloned into an appropriate expressionvector. Suitable humanized antibodies can alternatively be produced byCDR substitution U.S. Pat. No. 5,225,539; Jones (1986) Nature321:552-525; Verhoeyan et al, 1988 Science 239:1534; and Beidler (1988)J. Immunol. 141:4053-4060.

All of the CDRs of a particular human antibody may be replaced with atleast a portion of a non-human CDR or only some of the CDRs may bereplaced with non-human CDRs. It is only necessary to replace the numberof CDRs required for binding of the humanized antibody to the Fereceptor. An antibody can be humanized by any method, which is capableof replacing at least a portion of a CDR of a human antibody with a CDRderived from a non-human antibody. Winter describes a method which maybe used to prepare the humanized antibodies of the present invention,see UK Patent Application GB 2188638A, filed on Mar. 26, 1987. The humanCDRs may be replaced with non-human CDRs using oligonucleotidesite-directed mutagenesis as described in, e.g., WO 94/10332 entitled,Humanized Antibodies to Fc Receptors for Immunoglobulin G on HumanMononuclear Phagocytes.

Chimeric and humanized antibodies in which specific amino acids havebeen substituted, deleted or added are also within the scope of theinvention. For example, humanized antibodies can have amino acidsubstitutions in the framework region, such as to improve binding to theantigen. In a humanized antibody having mouse CDRs, amino acids locatedin the human framework region can be replaced with the amino acidslocated at the corresponding positions in the mouse antibody. Suchsubstitutions are known to improve binding of humanized antibodies tothe antigen in some instances. Antibodies in which amino acids have beenadded, deleted, or substituted are referred to herein as modifiedantibodies or altered antibodies.

Bispecific and multispecific molecules of the invention can furtherinclude a third binding specificity, in addition to an anti-Fc bindingspecificity and an anti-human CTLA-4 binding specificity. The thirdbinding specificity can be an anti-enhancement factor (BF) portion,e.g., a molecule which binds to a surface protein involved in cytotoxicactivity and thereby increases the immune response against the targetcell. The “anti-enhancement factor portion” can be an antibody,functional antibody fragment or a ligand that binds to a given molecule,e.g., an antigen or a receptor, and thereby results in an enhancement ofthe effect of the binding determinants for the Fc receptor or targetcell antigen. The “anti-enhancement factor portion” can bind an Fcreceptor or a target cell antigen. Alternatively, the anti-enhancementfactor portion can bind to an entity that is different from the entityto which the first and second binding specificities bind. For example,the anti-enhancement factor portion can bind a cytotoxic T-cell via,e.g., CD2, CD3, CD8, CD28, CD4, CD40, ICAM-1 or other immune cellmolecules that are involved in an increased immune response against thetarget cell.

Bispecific and multispecific molecules of the present invention can bemade using chemical techniques (see, e.g., Kranz (1981) Proc. Natl.Acad. Sci. USA 78:5807), “polydoma” techniques (see, e.g., U.S. Pat. No.4,474,893), or recombinant DNA techniques. Bispecific and multispecificmolecules of the present invention can also be prepared by conjugatingthe constituent binding specificities, e.g., the anti-FcR and anti-humanCTLA-4 binding specificities, using methods known in the art and asdescribed herein. For example, each binding specificity of thebispecific and multispecific molecule can be generated separately andthen conjugated to one another. When the binding specificities areproteins or peptides, a variety of coupling or cross-linking agents canbe used for covalent conjugation. Examples of cross-linking agentsinclude protein A, carbodiimide, N-succinimidyl-5-acetyl-thioacetate(SATA), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), andsulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate(sulfo-SMCC) (see, e.g., Karpovsky (1984) J. Exp. Med. 160:1686; Liu(1985) Proc. Natl. Acad. Sci. USA 82:8648). Other methods include thosedescribed by Paulus (Behring Ins. Mitt. (1985) No. 78, 118-132; Brennan(1985) Science 229:81-83), Glennie (1987) J. Immunol. 139: 2367-2375).Other conjugating agents are SATA and sulfo-SMCC, both available fromPierce Chemical Co. (Rockford, Ill.).

When the binding specificities are antibodies (e.g., two humanizedantibodies), they can be conjugated via sulfhydryl bonding of theC-terminus hinge regions of the two heavy chains. The hinge region canbe modified to contain an odd number of sulfhydryl residues, e.g., one,prior to conjugation.

Alternatively, both binding specificities can be encoded in the samevector and expressed and assembled in the same host cell. This method isparticularly useful where the bispecific and multispecific molecule is aMAb×MAb, MAb×Fab, Fab×F(ab′)₂ or ligand×Fab fusion protein. A bispecificand multispecific molecule of the invention, e.g., a bispecific moleculecan be a single chain molecule, such as a single chain bispecificantibody, a single chain bispecific molecule comprising one single chainantibody and a binding determinant, or a single chain bispecificmolecule comprising two binding determinants. Bispecific andmultispecific molecules can also be single chain molecules or maycomprise at least two single chain molecules. Methods for preparing bi-and multispecific molecules are described for example in U.S. Pat. No.5,260,203; U.S. Pat. No. 5,455,030; U.S. Pat. No. 4,881,175; U.S. Pat.No. 5,132,405; U.S. Pat. No. 5,091,513; U.S. Pat. No. 5,476,786; U.S.Pat. No. 5,013,653; U.S. Pat. No. 5,258,498; and U.S. Pat. No.5,482,858.

Binding of the bispecific and multispecific molecules to their specifictargets can be confirmed by enzyme-linked immunosorbent assay (ELISA), aradioimmunoassay (RIA), or a Western Blot Assay. Each of these assaysgenerally detects the presence of protein-antibody complexes ofparticular interest by employing a labeled reagent (e.g., an antibody)specific for the complex of interest. For example, the FcR-antibodycomplexes can be detected using e.g., an enzyme-linked antibody orantibody fragment which recognizes and specifically binds to theantibody-FcR complexes. Alternatively, the complexes can be detectedusing any of a variety of other immunoassays. For example, the antibodycan be radioactively labeled and used in a radioimmunoassay (RIA) (see,for example, Weintraub, B., Principles of Radioimmunoassays, SeventhTraining Course on Radioligand Assay Techniques, The Endocrine Society,March, 1986, which is incorporated by reference herein). The radioactiveisotope can be detected by such means as the use of a γ counter or ascintillation counter or by autoradiography.

Also included in the invention are modified antibodies. The term“modified antibody” includes antibodies, such as monoclonal antibodies,chimeric antibodies, and humanized antibodies which have been modifiedby, e.g., deleting, adding, or substituting portions of the antibody.For example, an antibody can be modified by deleting the constant regionand replacing it with a constant region meant to increase half-life,e.g., serum half-life, stability or affinity of the antibody.

The antibody conjugates of the invention can be used to modify a givenbiological response or create a biological response (e.g., to recruiteffector cells). The drug moiety is not to be construed as limited toclassical chemical therapeutic agents. For example, the drug moiety maybe a protein or polypeptide possessing a desired biological activity.Such proteins may include, for example, an enzymatically active toxin,or active fragment thereof, such as abrin, ricin A, pseudomonasexotoxin, or diphtheria toxin; a protein such as tumor necrosis factoror interferon-alpha; or, biological response modifiers such as, forexample, lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”),interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor(“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or othergrowth factors.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et cd. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev., 62:119-58 (1982).

Pharmaceutical Compositions

The invention provides pharmaceutical compositions comprising one or acombination of human monoclonal antibodies and/or human sequenceantibodies (intact or binding fragments) formulated together with apharmaceutically acceptable carrier. Some compositions include acombination of multiple (e.g., two or more) isolated human antibodiesand/or human sequence antibody or antigen-binding portions thereof ofthe invention. In some compositions, each of the antibodies orantigen-binding portions thereof of the composition is a monoclonalantibody or a human sequence antibody that binds to a distinct,pre-selected epitope of human CTLA-4.

A. Effective Dosages

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

Examples of pharmaceutically-acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention can be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level depends upon a variety of pharmacokinetic factors includingthe activity of the particular compositions of the present inventionemployed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors.

A physician or veterinarian can start doses of the compounds of theinvention employed in the pharmaceutical composition at levels lowerthan that required to achieve the desired therapeutic effect andgradually increase the dosage until the desired effect is achieved. Ingeneral, a suitable daily dose of a compositions of the invention isthat amount of the compound which is the lowest dose effective toproduce a therapeutic effect. Such an effective dose generally dependsupon the factors described above. It is preferred that administration beintravenous, intramuscular, intraperitoneal, or subcutaneous, oradministered proximal to the site of the target. If desired, theeffective daily dose of a therapeutic compositions can be administeredas two, three, four, five, six or more sub-doses administered separatelyat appropriate intervals throughout the day, optionally, in unit dosageforms. While it is possible for a compound of the present invention tobe administered alone, it is preferable to administer the compound as apharmaceutical formulation (composition).

Effective doses of the compositions of the present invention, for thetreatment of immune-related conditions and diseases described hereinvary depending upon many different factors, including means ofadministration, target site, physiological state of the patient, whetherthe patient is human or an animal, other medications administered, andwhether treatment is prophylactic or therapeutic. Treatment dosages needto be titrated to optimize safety and efficacy.

For administration with an antibody, the dosage ranges from about 0.0001to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight.For example dosages can be 1 mg/kg body weight or 10 mg/kg body weightor within the range of 1-10 mg/kg. An exemplary treatment regime entailsadministration once per every two weeks or once a month or once every 3to 6 months. In some methods, two or more monoclonal antibodies withdifferent binding specificities are administered simultaneously, inwhich case the dosage of each antibody administered falls within theranges indicated. Antibody is usually administered on multipleoccasions. Intervals between single dosages can be weekly, monthly oryearly. Intervals can also be irregular as indicated by measuring bloodlevels of antibody to CTLA-4 in the patient. In some methods, dosage isadjusted to achieve a plasma antibody concentration of 1-1000 μg/ml andin some methods 25-300 μg/ml. Alternatively, antibody can beadministered as a sustained release formulation, in which case lessfrequent administration is required. Dosage and frequency vary dependingon the half-life of the antibody in the patient. In general, humanantibodies show the longest half life, followed by humanized antibodies,chimeric antibodies, and nonhuman antibodies. The dosage and frequencyof administration can vary depending on whether the treatment isprophylactic or therapeutic. In prophylactic applications, a relativelylow dosage is administered at relatively infrequent intervals over along period of time. Some patients continue to receive treatment for therest of their lives. In therapeutic applications, a relatively highdosage at relatively short intervals is sometimes required untilprogression of the disease is reduced or terminated, and preferablyuntil the patient shows partial or complete amelioration of symptoms ofdisease. Thereafter, the patent can be administered a prophylacticregime.

Doses for nucleic acids encoding immunogens range from about 10 ng to 1g, 100 ng to 100 mg, 1 μg to 10 mg, or 30-300 μg DNA per patient. Dosesfor infectious viral vectors vary from 10-100, or more, virions perdose.

Some human sequence antibodies and human monoclonal antibodies of theinvention can be formulated to ensure proper distribution in vivo. Forexample, the blood-brain barrier (BBB) excludes many highly hydrophiliccompounds. To ensure that the therapeutic compounds of the inventioncross the BBB (if desired), they can be formulated, for example, inliposomes. For methods of manufacturing liposomes, See, e.g., U.S. Pat.Nos. 4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise oneor more moieties which are selectively transported into specific cellsor organs, thus enhance targeted drug delivery (See, e.g., V. V. Ranade(1989) J. Clin. Pharmacal. 29:685). Exemplary targeting moieties includefolate or biotin (See, e.g., U.S. Pat. No. 5,416,016 to Low et al.);mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun.153:1038); antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357:140;M. Owais et al., (1995) Antimicrob. Agents Chemother. 39:180);surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol.1233:134), different species of which may comprise the formulations ofthe inventions, as well as components of the invented molecules; p120(Schreier et al. (1994) J. Biol. Chem. 269:9090); See also K. Keinanen;M. L. Laukkanen (1994) FEBS Lett. 346:123; J. J. Killion; I. J. Fidler(1994) Immunomethods 4:273. In some methods, the therapeutic compoundsof the invention are formulated in liposomes; in a more preferredembodiment, the liposomes include a targeting moiety. In some methods,the therapeutic compounds in the liposomes are delivered by bolusinjection to a site proximal to the tumor or infection. The compositionshould be fluid to the extent that easy syringability exists. It shouldbe stable under the conditions of manufacture and storage and should bepreserved against the contaminating action of microorganisms such asbacteria and fungi.

For therapeutic applications, the pharmaceutical compositions areadministered to a patient suffering from established disease in anamount sufficient to arrest or inhibit further development or reverse oreliminate, the disease, its symptoms or biochemical markers. Forprophylactic applications, the pharmaceutical compositions areadministered to a patient susceptible or at risk of a disease in anamount sufficient to delay, inhibit or prevent development of thedisease, its symptoms and biochemical markers. An amount adequate toaccomplish this is defined as a “therapeutically-” or“prophylactically-effective dose.” Dosage depends on the disease beingtreated, the subject's size, the severity of the subject's symptoms, andthe particular composition or route of administration selected.Specifically, in treatment of tumors, a “therapeutically effectivedosage” can inhibit tumor growth by at least about 20%, or at leastabout 40%, or at least about 60%, or at least about 80% relative tountreated subjects. The ability of a compound to inhibit cancer can beevaluated in an animal model system predictive of efficacy in humantumors. Alternatively, this property of a composition can be evaluatedby examining the ability of the compound to inhibit by conventionalassays in vitro. A therapeutically effective amount of a therapeuticcompound can decrease tumor size, or otherwise ameliorate symptoms in asubject.

The composition should be sterile and fluid to the extent that thecomposition is deliverable by syringe. In addition to water, the carriercan be an isotonic buffered saline solution, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyetheylene glycol,and the like), and suitable mixtures thereof. Proper fluidity can bemaintained, for example, by use of coating such as lecithin, bymaintenance of required particle size in the case of dispersion and byuse of surfactants. In many cases, it is preferable to include isotonicagents, for example, sugars, polyalcohols such as mannitol or sorbitol,and sodium chloride in the composition. Long-term absorption of theinjectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, aluminummonostearate or gelatin.

When the active compound is suitably protected, as described above, thecompound may be orally administered, for example, with an inert diluentor an assimilable edible carrier.

B. Routes of Administration

Pharmaceutical compositions of the invention also can be administered incombination therapy, i.e., combined with other agents. For example, intreatment of cancer, the combination therapy can include a compositionof the present invention with at least one anti-tumor agent or otherconventional therapy, such as radiation treatment.

Pharmaceutically acceptable carriers includes solvents, dispersionmedia, coatings, antibacterial and antifungal agents, isotonic andabsorption delaying agents, and the like that are physiologicallycompatible. The carrier can be suitable for intravenous, intramuscular,subcutaneous, parenteral, spinal or epidermal administration (e.g., byinjection or infusion). Depending on the route of administration, theactive compound, i.e., antibody, bispecific and multispecific molecule,may be coated in a material to protect the compound from the action ofacids and other natural conditions that may inactivate the compound.

A “pharmaceutically acceptable salt” refers to a salt that retains thedesired biological activity of the parent compound and does not impartany undesired toxicological effects (See, e.g., Berge, S. M., et al.(1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acidaddition salts and base addition salts. Acid addition salts includethose derived from nontoxic inorganic acids, such as hydrochloric,nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous andthe like, as well as from nontoxic organic acids such as aliphatic mono-and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acidsand the like. Base addition salts include those derived from alkalineearth metals, such as sodium, potassium, magnesium, calcium and thelike, as well as from nontoxic organic amines, such asN,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,choline, diethanolamine, ethylenediamine, procaine and the like.

A composition of the present invention can be administered by a varietyof methods known in the art. The route and/or mode of administrationvary depending upon the desired results. The active compounds can beprepared with carriers that protect the compound against rapid release,such as a controlled release formulation, including implants,transdermal patches, and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Many methods for the preparationof such formulations are described by e.g., Sustained and ControlledRelease Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc.,New York, 1978. Pharmaceutical compositions are preferably manufacturedunder GMP conditions.

To administer a compound of the invention by certain routes ofadministration, it may be necessary to coat the compound with, orco-administer the compound with, a material to prevent its inactivation.For example, the compound may be administered to a subject in anappropriate carrier, for example, liposomes, or a diluent.Pharmaceutically acceptable diluents include saline and aqueous buffersolutions. Liposomes include water-in-oil-in-water CGF emulsions as wellas conventional liposomes (Strejan et al. (1984) J. Neuroimmunol. 7:27).

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofthe invention is contemplated. Supplementary active compounds can alsobe incorporated into the compositions.

Therapeutic compositions typically must be sterile, substantiallyisotonic, and stable under the conditions of manufacture and storage.The composition can be formulated as a solution, microemulsion,liposome, or other ordered structure suitable to high drugconcentration. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyethylene glycol, and the like), andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. In many cases, it is preferable to include isotonic agents,for example, sugars, polyalcohols such as mannitol, sorbitol, or sodiumchloride in the composition. Prolonged absorption of the injectablecompositions can be brought about by including in the composition anagent that delays absorption, for example, monostearate salts andgelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof. Therapeutic compositionscan also be administered with medical devices known in the art. Forexample, in a preferred embodiment, a therapeutic composition of theinvention can be administered with a needleless hypodermic injectiondevice, such as the devices disclosed in, e.g., U.S. Pat. No. 5,399,163,5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556.Examples of implants and modules useful in the present inventioninclude: U.S. Pat. No. 4,487,603, which discloses an implantablemicro-infusion pump for dispensing medication at a controlled rate; U.S.Pat. No. 4,486,194, which discloses a therapeutic device foradministering medicants through the skin; U.S. Pat. No. 4,447,233, whichdiscloses a medication infusion pump for delivering medication at aprecise infusion rate; U.S. Pat. No. 4,447,224, which discloses avariable flow implantable infusion apparatus for continuous drugdelivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drugdelivery system having multi-chamber compartments; and U.S. Pat. No.4,475,196, which discloses an osmotic drug delivery system. Many othersuch implants, delivery systems, and modules are known.

C. Formulation

For the therapeutic compositions, formulations of the present inventioninclude those suitable for oral, nasal, topical (including buccal andsublingual), rectal, vaginal and/or parenteral administration. Theformulations can conveniently be presented in unit dosage form and maybe prepared by any methods known in the art of pharmacy. The amount ofactive ingredient which can be combined with a carrier material toproduce a single dosage form vary depending upon the subject beingtreated, and the particular mode of administration. The amount of activeingredient which can be combined with a carrier material to produce asingle dosage form generally be that amount of the composition whichproduces a therapeutic effect. Generally, out of one hundred percent,this amount range from about 0.01 percent to about ninety-nine percentof active ingredient, from about 0.1 percent to about 70 percent, orfrom about 1 percent to about 30 percent.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate. Dosage forms for the topical or transdermaladministration of compositions of this invention include powders,sprays, ointments, pastes, creams, lotions, gels, solutions, patches andinhalants. The active compound may be mixed under sterile conditionswith a pharmaceutically acceptable carrier, and with any preservatives,buffers, or propellants which may be required.

The phrases “parenteral administration” and “administered parenterally”mean modes of administration other than enteral and topicaladministration, usually by injection, and includes, without limitation,intravenous, intramuscular, intraarterial, intrathecal, intracapsular,intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal,subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid,intraspinal, epidural and intrasternal injection and infusion.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

When the compounds of the present invention are administered aspharmaceuticals, to humans and animals, they can be given alone or as apharmaceutical composition containing, for example, 0.01 to 99.5% (or0.1 to 90%) of active ingredient in combination with a pharmaceuticallyacceptable carrier.

The pharmaceutical compositions are generally formulated as sterile,substantially isotonic and in full compliance with all GoodManufacturing Practice (GMP) regulations of the U.S. Food and DrugAdministration.

Methods and Uses of the Invention

A. Methods

The compositions (e.g., human sequence antibodies and human monoclonalantibodies to human CTLA-4 and derivatives/conjugates thereof) of thepresent invention have in vitro and in vivo diagnostic and therapeuticutilities. For example, these molecules can be administered to cells inculture, e.g. in vitro or ex vivo, or in a subject, e.g., in vivo, totreat, prevent or diagnose a variety of disorders. The term “subject”includes human and non-human animals. Non-human animals includes allvertebrates, e.g., mammals and non-mammals, such as non-human primates,sheep, dog, cow, chickens, amphibians, and reptiles. The methods areparticularly suitable for treating human patients having a disorder thatcan be treated by augmenting or reducing the T-cell mediated immuneresponse.

When antibodies to CTLA-4 are administered together with another agent,the two can be administered in either order or simultaneously. Themethods can be used to treat any kind of cancer including melanoma,colon cancer, prostate cancer, and renal cancer.

For example, latex microspheres coated with anti-CTLA-4 (to increase thevalency of the antibody) can inhibit T cell proliferation andactivation. Agents having the same antibody combining site may act as aCTLA-4 antagonist when presented as an Fab or a soluble IgG, and aCTLA-4 agonist when highly cross-linked. Thus multivalent forms ofanti-CTLA-4 antibodies are useful therapeutic agents for down-modulatingimmune responses.

In addition to linking to latex microspheres or other insolubleparticles, the antibodies can be cross-linked to each other orgenetically engineered to form multimers. Cross-linking can be by directchemical linkage, or by indirect linkage such as anantibody-biotin-avidin complex. Cross-linking can be covalent, wherechemical linking groups are employed, or non-covalent, whereprotein-protein or other protein-ligand interactions are employed.Genetic engineering approaches for linking include, e.g., there-expression of the variable regions of high-affinity IgG antibodies inIgM expression vectors or any protein moiety (e.g., polylysine, and thelike). Converting a high affinity IgG antibody to an IgM antibody cancreate a decavalent complex with very high avidity. IgA₂ expressionvectors may also be used to produce multivalent antibody complexes. IgA₂can form polymers together with J chain and secretory component. IgA₂may have the added advantage that it can be additionally crosslinked bythe IgA receptor CD89, which is expressed on neutrophils, macrophages,and monocytes.

Agonism can also be obtained using some preparations of polyclonalantibodies to CTLA-4 comprising antibodies to at least twonon-overlapping epitopes on CTLA-4. One antibody in such a preparationcontaining two binding sites can bind to two molecules of CTLA-4 to forma small cluster. A second antibody possessing different binding sitescan then link (aggregate) these small clusters to form large clusters,thereby forming a complex of CTLA-4 (on the cell surface) that cantransduce a signal to the T cell to inhibit, reduce or preventactivation of the T-cell bearing (expressing) CTLA-4. Thus, somepreparations of polyclonal antibodies show similar agonism to thepolyvalent preparations described above.

Therefore, polyvalent or polyclonal preparations of anti CTLA-4antibodies are useful for agonizing the CTLA-4 receptor, therebysuppressing immune responses otherwise mediated by T cells bearing theCTLA-4 receptor. Some examples of diseases that can be treated usingsuch polyvalent or polyclonal preparations of antibodies induceautoimmune disease, transplant rejection, and inflammation.

B. Uses

1. Activating Immune Responses

a. Cancer

Some therapeutic methods treat patients with cancer. Blockade of CTLA-4by antibodies can enhance the immune response to cancerous cells in thepatient. Optionally, antibodies to CTLA-4 can be combined with animmunogenic agent, such as cancerous cells, purified tumor antigens(including recombinant proteins, peptides, and carbohydrate molecules),cells, and cells transfected with genes encoding immune stimulatingcytokines and cell surface antigens such as B7 (see, e.g., Hurwitz, A.et al. (1998) Proc. Natl. Acad. Sci. USA. 95, 10067-10071).

In murine experimental systems, implantation of some tumors followed bythe administration of anti-CTLA-4 antibodies can result in the rejectionof tumors. In some cases tumor rejection of established tumors occurs;in other cases the growth of the tumor is slowed by the use ofanti-CTLA-4 antibodies. In general CTLA-4 blockade is effective againstimmunogenic tumors. Operationally this is defined as those tumors forwhich vaccination using the tumor itself can lead to immunity to tumorchallenge. In humans, some tumors have been shown to be immunogenic suchas melanomas. It is anticipated that by raising the threshold of T cellactivation by CTLA-4 blockade, we may expect to activate tumor responsesin the host.

CTLA-4 blockade is most effective when combined with a vaccinationprotocol. Many experimental strategies for vaccination against tumorshave been devised (see Rosenberg, S., 2000, Development of CancerVaccines, ASCO Educational Book Spring: 60-62; Logothetis, C., 2000,ASCO Educational Book Spring: 300-302; Khayat, D. 2000, ASCO EducationalBook Spring: 414-428; Foon, K. 2000, ASCO Educational Book Spring:730-738; see also Restifo, N. and Sznol, M., Cancer Vaccines, Ch. 61,pp. 3023-3043 in DeVita, V. et al. (eds.), 1997, Cancer: Principles andPractice of Oncology, Fifth Edition). In one of these strategies, avaccine is prepared using autologous or allogeneic tumor cells. Thesecellular vaccines have been shown to be most effective when the tumorcells are transduced to express GM-CSF. GM-CSF has been shown to be apotent activator of antigen presentation for tumor vaccination (Dranoffet al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90 (80: 3539-43).

Anti-CTLA-4 blockade together with the use of GMCSF-modified tumor cellvaccines has been shown to be effective in a number of experimentaltumor models such as mammary carcinoma (Hurwitz et al. (1998) supra),primary prostate cancer (Hurwitz A. et al., (2000) Cancer Research 60(9): 2444-8) and melanoma (van Elsas, A et al. (1999) J. Exp. Med. 190:355-66). In these instances, non-immunogenic tumors, such as the B16melanoma, have been rendered susceptible to destruction by the immunesystem. The tumor cell vaccine may also be modified to express otherimmune activators such as IL2, and costimulatory molecules, amongothers.

The study of gene expression and large scale gene expression patterns invarious tumors has led to the definition of so called tumor specificantigens (Rosenberg, S A (1999) Immunity 10: 281-7). In many cases,these tumor specific antigens are differentiation antigens expressed inthe tumors and in the cell from which the tumor arose, for examplemelanocyte antigens gp 100, MAGE antigens, Trp-2. More importantly, manyof these antigens can be shown to be the targets of tumor specific Tcells found in the host. CTLA-4 blockade may be used in conjunction witha collection of recombinant proteins and/or peptides expressed in atumor in order to generate an immune response to these proteins. Theseproteins are normally viewed by the immune system as self antigens andare therefore tolerant to them. The tumor antigen may also include theprotein telomerase, which is required for the synthesis of telomeres ofchromosomes and which is expressed in more than 85% of human cancers andin only a limited number of somatic tissues (Kim, N et al. (1994)Science 266, 2011-2013). (These somatic tissues may be protected fromimmune attack by various means). Tumor antigen may also be“neo-antigens” expressed in cancer cells because of somatic mutationsthat alter protein sequence or create fusion proteins between twounrelated sequences (ie. bcr-abl in the Philadelphia chromosome), oridiotype from B cell tumors. Other tumor vaccines may include theproteins from viruses implicated in human cancers such a Human PapillomaViruses (HPV), Hepatitis Viruses (HBV and HCV) and Kaposi's HerpesSarcoma Virus (KHSV). Another form of tumor specific antigen which maybe used in conjunction with CTLA-4 blockade is purified heat shockproteins (HSP) isolated from the tumor tissue itself. These heat shockproteins contain fragments of proteins from the tumor cells and theseHSPs are highly efficient at delivery to antigen presenting cells foreliciting tumor immunity (Suot, R & Srivastava, P (1995) Science 269:1585-1588; Tamura, Y. et al., (1997) Science 278: 117-120.

Dendritic cells (DC) are potent antigen presenting cells that can beused to prime antigen-specific responses. DC's can be produced ex vivoand loaded with various protein and peptide antigens as well as tumorcell extracts (Nestle, F. et al. (1998) Nature Medicine 4: 328-332). DCsmay also be transduced by genetic means to express these tumor antigensas well. DCs have also been fused directly to tumor cells for thepurposes of immunization (Kugler, A. et al. (2000) Nature Medicine6:332-336). As a method of vaccination, DC immunization may beeffectively combined with CTLA-4 blockade to activate more potentanti-tumor responses.

CTLA-4 blockade may also be combined with standard cancer treatments.CTLA-4 blockade may be effectively combined with chemotherapeuticregimes. In these instances, it may be possible to reduce the dose ofchemotherapeutic reagent administered (Mokyr, M. et al. (1998) CancerResearch 58: 5301-5304). The scientific rationale behind the combineduse of CTLA-4 blockade and chemotherapy is that cell death, that is aconsequence of the cytotoxic action of most chemotherapeutic compounds,should result in increased levels of tumor antigen in the antigenpresentation pathway. Other combination therapies that may result insynergy with CTLA-4 blockade through cell death are radiation, surgery,and hormone deprivation (Kwon, E. et al. (1999) Proc. Natl. Acad. Sci.U.S.A. 96 (26): 15074-9. Each of these protocols creates a source oftumor antigen in the host. Angiogenesis inhibitors may also be combinedwith CTLA-4 blockade. Inhibition of angiogenesis leads to tumor celldeath which may feed tumor antigen into host antigen presentationpathways.

CTLA-4 blocking antibodies can also be used in combination withbispecific antibodies that target Fe alpha or Fc gammareceptor-expressing effectors cells to tumor cells (see, e.g., U.S. Pat.Nos. 5,922,845 and 5,837,243). Bispecific antibodies can be used totarget two separate antigens. For example anti-Fc receptor/anti tumorantigen (i.e., Her-2/neu) bispecific antibodies have been used to targetmacrophages to sites of tumor. This targeting may more effectivelyactivate tumor specific responses. The T cell arm of these responseswould by augmented by the use of CTLA-4 blockade. Alternatively, antigenmay be delivered directly to DCs by the use of bispecific antibodieswhich bind to tumor antigen and a dendritic cell specific cell surfacemarker.

Tumors evade host immune surveillance by a large variety of mechanisms.Many of these mechanisms may be overcome by the inactivation of proteinswhich are expressed by the tumors and which are immunosuppressive. Theseinclude among others Tgfβ (Kehrl, J. et al. (1986) J. Exp. Med. 163:1037-1050), IL-10 (Howard, M. & O'Garra, A. (1992) Immunology Today 13:198-200), and Fas ligand (Hahne, M. et al. (1996) Science 274:1363-1365). Antibodies to each of these entities may be used incombination with anti-CTLA-4 to counteract the effects of theimmunosuppressive agent and favor tumor immune responses by the host.

Other antibodies which may be used to activate host immuneresponsiveness can be used in combination with anti-CTLA-4. Theseinclude molecules on the surface of dendritic cells which activate DCfunction and antigen presentation. Anti-CD40 antibodies are able tosubstitute effectively for T cell helper activity (Ridge, J. et al.(1998) Nature 393: 474-478). and can be used in conjunction with CTLA-4antibodies (Ito, N. et al. (2000) Immunobiology 201 (5) 527-40).Activating antibodies to T cell costimulatory molecules such as OX-40(Weinberg, A. et al. (2000) J Immunol 164: 2160-2169), 4-1BB (Melero, I.et al. (1997) Nature Medicine 3: 682-685 (1997), and ICOS (Hutloff, A.et al. (1999) Nature 397: 262-266) may also provide for increased levelsof T cell activation.

Bone marrow transplantation is currently being used to treat a varietyof tumors of hematopoietic origin. While graft versus host disease is aconsequence of this treatment, therapeutic benefit may be obtained fromgraft vs. tumor responses. CTLA-4 blockade can be used to increase theeffectiveness of the donor engrafted tumor specific T cells (Blazar, B.et al. (1999) J Immunol 162: 6368-6377).

There are also several experimental treatment protocols that involve exvivo activation and expansion of antigen specific T cells and adoptivetransfer of these cells into recipients in order to antigen-specific Tcells against tumor (Greenberg, R. & Riddell, S. (1999) 285: 546-51).These methods may also be used to activate T cell responses toinfectious agents such as CMV (see below). Ex vivo activation in thepresence of anti-CTLA-4 antibodies may be expected to increase thefrequency and activity of the adoptively transferred T cells.

b. Infectious Diseases

Other methods of the invention are used to treat patients that have beenexposed to particular toxins or pathogens. Similar to its application totumors as discussed above, antibody mediated CTLA-4 blockade can be usedalone, or as an adjuvant, in combination with vaccines, to stimulate theimmune response to pathogens, toxins, and self-antigens. CTLA-4 blockadehas been shown to be effective in the acute phase of infections ofNippostrongylus brasiliensis (McCoy, K. et al. (1997) 186(2); 183-187)and Leishmania donovani (Murphy, M. et al. (1998) J. Immunol.161:4153-4160). Examples of pathogens for which this therapeuticapproach may be particularly useful, include pathogens for which thereis currently no effective vaccine, or pathogens for which conventionalvaccines are less than completely effective. These include, but are notlimited to HIV, Hepatitis (A, B, & C), Influenza, Herpes, Giardia,Malaria, Leishmania, Staphylococcus Aureus, Pseudomonas aeruginosa.CTLA-4 blockade is particularly useful against established infections byagents such as HIV that present altered antigens over the course of theinfections. These novel epitopes are recognized as foreign at the timeof anti-human CTLA-4 administration, thus provoking a strong T cellresponse that is not dampened by negative signals through CTLA-4.

Some examples of pathogenic viruses causing infections treatable bymethods of the invention include hepatitis (A, B, or C), herpes virus(e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein Barr virus),adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus,coxsackie virus, cornovirus, respiratory syncytial virus, mumps virus,rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus,HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus,rabies virus, JC virus and arboviral encephalitis virus.

Some examples of pathogenic bacteria causing infections treatable bymethods of the invention include chlamydia, rickettsial bacteria,mycobacteria, staphylococci, streptococci, pneumonococci, meningococciand conococci, klebsiella, proteus, serratia, pseudomonas, legionella,diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax,plague, leptospirosis, and Lymes disease bacteria.

Some examples of pathogenic fungi causing infections treatable bymethods of the invention include Candida (albicans, krusei, glabrata,tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus,niger, etc.), Genus Mucorales (Mucor, Absidia, Rhizophus), Sporothrixschenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis,Coccidioides immitis and Histoplasma capsulatum.

Some examples of pathogenic parasites causing infections treatable bymethods of the invention include Entamoeba histolytica, Balantidiumcoli, Naegleria fowleri, Acanthamoeba sp., Giardia lambia,Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesiamicroti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani,Toxoplasma gondi, Nippostrongylus brasiliensis.

In all of the above methods, a CTLA-4 blockade can be combined withother forms of immunotherapy such as cytokine treatment (e.g.interferons, GM-CSF, GCSF, IL-2), or bispecific antibody therapy, whichprovides for enhanced presentation of tumor antigens (see, e.g.,Holliger (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak (1994)Structure 2:1121-1123).

c. Promoting Beneficial “Autoimmune” Reactions for the Treatment ofDisease and Therapeutic Intervention.

The ability of anti-CTLA-4 antibodies to provoke and amplify autoimmuneresponses has been documented in a number of experimental systems(EAE—Experimental Autoimmune Encephalomyelitis, a murine model for MS(Perrin, P. et al. (1996) J Immunol 157 (4): 1333-1336); diabetes(Luhder, F. et al. (1998) supra). Indeed, induction of anti-tumorresponses using tumor cell and peptide vaccines reveals that manyanti-tumor responses involve anti-self reactivities (depigmentationobserved in anti-CTLA-4+GM-CSF modified B16 melanoma in van Elsas et al.supra; depigmentation in Trp-2 vaccinated mice (Overwijk, W. et al.(1999) Proc. Natl. Acad. Sci. U.S.A. 96: 2982-2987); autoimmuneprostatitis evoked by TRAMP tumor cell vaccines (Hurwitz, A. (2000)supra), melanoma peptide antigen vaccination and vitilago observed inhuman clinical trials (Rosenberg, S A and White, D E (1996) J ImmunotherEmphasis Tumor Immunol 19 (1): 81-4).

Therefore, it is possible to consider using anti-CTLA-4 blockade inconjunction with various self proteins in order to devise vaccinationprotocols to efficiently generate immune responses against these selfproteins for disease treatment. For example, Alzheimers disease involvesinappropriate accumulation of Aβ peptide in amyloid deposits in thebrain; antibody responses against amyloid are able to clear theseamyloid deposits (Schenk et al., (1999) Nature 400: 173-177).

Other self proteins may also be used as targets such as IgE for thetreatment of allergy and asthma, and TNF for rhematoid arthritis.Finally, antibody responses to various hormones may be induced by theuse of anti-CTLA-4 antibody. Neutralizing antibody responses toreproductive hormones may be used for contraception. Neutralizingantibody response to hormones and other soluble factors that arerequired for the growth of particular tumors may also be considered aspossible vaccination targets.

Analogous methods as described above for the use of anti-CTLA-4 antibodycan be used for induction of therapeutic autoimmune responses to treatpatients having an inappropriate accumulation of other self-antigens,such as amyloid deposits, including Aβ in Alzheimer's disease, cytokinessuch as TNFα, and IgE.

2. Inactivating Immune Responses

Disorders caused by immune responses are called hypersensitivitydisease. Diseases caused by failure of self-tolerance and subsequentimmune responses against self, or autologous, antigens are calledautoimmune diseases. Hypersensitivity diseases can also result fromuncontrolled or excessive responses against foreign antigens, such asmicrobes.

Although soluble antibodies to human CTLA-4 have been shown to promotethe expansion and activation of T cells (i.e., where CTLA-4 function(e.g., binding to ligand) is inhibited; in this scenario the antibodiesare antagonists to CTLA-4 function), increasing the valency of thesesame antibodies produces the opposite effect (where now, in contrast,the antibodies are acting as agonists of CTLA-4 to suppress the immuneresponse) (see, e.g., Krummel and Allison, 1996, J. Exp. Med. 183,2533-2540). For the purposes of inactivating antigen specific T cellresponses, such as those that are the targets of pathogenic autoreactiveT cells, the target antigen which is specific for these T cells (i.e.antigen and/or MHC/antigen complexes) must be administered with thepolyvalent form of anti-CTLA-4 antibody.

a. Inflammation

Inflammation represents the consequence of capillary dilation withaccumulation of fluid and migration of phagocytic leukocytes, such asgranulocytes and monocytes. Inflammation is important in defending ahost against a variety of infections but can also have undesirableconsequences in inflammatory disorders, such as anaphylactic shock,arthritis, gout and ischemia-reperfusion. Activated T-cells have animportant modulatory role in inflammation, releasing interferon γ andcolony stimulating factors that in turn activate phagocytic leukocytes.The activated phagocytic leukocytes are induced to express a number ofspecific cells surface molecules termed homing receptors, which serve toattach the phagocytes to target endothelial cells. Inflammatoryresponses can be reduced or eliminated by treatment with the therapeuticagents of the present invention. For example, polyvalent preparations ofantibodies against CTLA-4 block activation of activated T-cells, therebypreventing these cells from releasing molecules required for activationof phagocytic cell types

b. Autoimmune Diseases

A further situation in which immune suppression is desirable is intreatment of autoimmune diseases such as insulin-dependent diabetesmellitus, multiple sclerosis, stiff man syndrome, rheumatoid arthritis,myasthenia gravis and lupus erythematosus. In these diseases, the bodydevelops a cellular and/or humoral immune response against one of itsown antigens leading to destruction of that antigen, and potentiallycrippling and/or fatal consequences. Activated T-cells are believed toplay a major role in many autoimmune diseases such as diabetes mellitus.Autoimmune diseases are treated by administering one of the therapeuticagents of the invention that inhibits activation of T cells. Optionally,the autoantigen, or a fragment thereof, against which the autoimmunedisease is targeted can be administered shortly before, concurrentlywith, or shortly after the immunosuppressive agent. In this manner,tolerance can be induced to the autoantigen under cover of thesuppressive treatment, thereby obviating the need for continuedimmunosuppression. See, e.g., Cobbold et al., WO 90/15152 (1990).

c. Graft Versus Host Disease

A related use for the therapeutic agents of the present invention is inmodulating the immune response involved in “graft versus host” disease(GVHD). GVHD is a potentially fatal disease that occurs whenimmunologically competent cells are transferred to an allogeneicrecipient. In this situation, the donor's immunocompetent cells mayattack tissues in the recipient. Tissues of the skin, gut epithelia andliver are frequent targets and may be destroyed during the course ofGVHD. The disease presents an especially severe problem when immunetissue is being transplanted, such as in bone marrow transplantation;but less severe GVHD has also been reported in other cases as well,including heart and liver transplants. The therapeutic agents of thepresent invention are used to inhibit activation of donor leukocytes,thereby inhibiting their ability to lyse target cells in the host.

d. Transplant Rejection

Over recent years there has been a considerable improvement in theefficiency of surgical techniques for transplanting tissues and organssuch as skin, kidney, liver, heart, lung, pancreas and bone marrow.Perhaps the principal outstanding problem is the lack of satisfactoryagents for inducing immune-tolerance in the recipient to thetransplanted allograft or organ. When allogeneic cells or organs aretransplanted into a host (i.e., the donor and donee are differentindividual from the same species), the host immune system is likely tomount an immune response to foreign antigens in the transplant(host-versus-graft disease) leading to destruction of the transplantedtissue. CD8⁺ cells, CD4⁺ cells and monocytes are all involved in therejection of transplant tissues. The therapeutic agents of the presentinvention are useful to inhibit T-cell mediated alloantigen-inducedimmune responses in the donee thereby preventing such cells fromparticipating in the destruction of the transplanted tissue or organ.

B. Methods for Detecting/Measuring the Presence of CTLA-4 in a Sample

The invention further provides methods for detecting the presence ofhuman CTLA-4 antigen in a sample, or measuring the amount of humanCTLA-4 antigen, comprising contacting the sample, and a control sample,with a human monoclonal antibody, or an antigen binding portion thereof,which specifically binds to human CTLA-4, under conditions that allowfor formation of a complex between the antibody or portion thereof andhuman CTLA-4. The formation of a complex is then detected, wherein adifference complex formation between the sample compared to the controlsample is indicative the presence of human CTLA-4 antigen in the sample.

C. Kits

Also within the scope of the invention are kits comprising thecompositions (e.g., human sequence antibodies, human antibodies,multispecific and bispecific molecules) of the invention andinstructions for use. The kit can further contain a least one additionalreagent, or one or more additional human antibodies of the invention(e.g., a human antibody having a complementary activity which binds toan epitope in CTLA-4 antigen distinct from the first human antibody).Kits typically include a label indicating the intended use of thecontents of the kit. The term label includes any writing, or recordedmaterial supplied on or with the kit, or which otherwise accompanies thekit.

EXAMPLES Example 1 Generation of Cmu Targeted Mice

Construction of a CMD Targeting Vector.

The plasmid pICEmu contains an EcoRI/XhoI fragment of the murine Igheavy chain locus, spanning the mu gene, that was obtained from a Balb/Cgenomic lambda phage library (Marco et al. Cell 22: 187, 1980). Thisgenomic fragment was subcloned into the XhoI/EcoRI sites of the plasmidpICEMI9H (Marsh et al.; Gene 32, 481-485, 1984). The heavy chainsequences included in pICEmu extend downstream of the EcoRI site locatedjust 3′ of the mu intronic enhancer, to the XhoI site locatedapproximately 1 kb downstream of the last transmembrane exon of the mugene; however, much of the mu switch repeat region has been deleted bypassage in E. coli.

The targeting vector was constructed as follows (see FIG. 1). A 1.3 kbHindIII/SmaI fragment was excised from pICEmu and subcloned intoHindIII/SmaI digested pBluescript (Stratagene, La Jolla, Calif.). ThispICEmu fragment extends from the HindIII site located approximately 1 kb5′ of CmuI to the SmaI site located within Cmu 1. The resulting plasmidwas digested with SmaI/SpeI and the approximately 4 kb SmaI/XbaIfragment from pICEmu, extending from the Sma I site in Cmu1 3′ to theXbaI site located just downstream of the last Cmu exon, was inserted.The resulting plasmid, pTAR1, was linearized at the SmaI site, and a neoexpression cassette inserted. This cassette consists of the neo geneunder the transcriptional control of the mouse phosphoglycerate kinase(pgk) promoter (XbaI/TaqI fragment; Adra et al. (1987) Gene 60: 65-74)and containing the pgk polyadenylation site (PvuII/HindIII fragment;Boer et al., (1990) Biochemical Genetics 28: 299-308). This cassette wasobtained from the plasmid pKJ1 (described by Tybulewicz et al. (1991)Cell 65: 1153-1163) from which the neo cassette was excised as anEcoRI/HindIII fragment and subcloned into EcoRI/HindIII digestedpGEM-7Zf (+) to generate pGEM-7 (KJ1). The neo cassette was excised frompGEM-7 (KJ1) by EcoRI/SalI digestion, blunt ended and subcloned into theSmaI site of the plasmid pTAR1, in the opposite orientation of thegenomic Cmu sequences. The resulting plasmid was linearized with Not I,and a herpes simplex virus thymidine kinase (tk) cassette was insertedto allow for enrichment of ES clones bearing homologous recombinants, asdescribed by Mansour et al., (1988) Nature 336: 348-352. This cassetteconsists of the coding sequences of the tk gene bracketed by the mousepgk promoter and polyadenylation site, as described by Tybulewicz et al.(1991) Cell 65: 1153-1163. The resulting CMD targeting vector contains atotal of approximately 5.3 kb of homology to the heavy chain locus andis designed to generate a mutant mu gene into which has been inserted aneo expression cassette in the unique SmaI site of the first Cmu exon.The targeting vector was linearized with PvuI, which cuts within plasmidsequences, prior to electroporation into ES cells.

Generation and Analysis of Targeted ES Cells.

AB-1 ES cells (McMahon, A. P. and Bradley, A., (1990) Cell 62:1073-1085) were grown on mitotically inactive SNL76/7 cell feeder layers(ibid.) essentially as described (Robertson, E. J. (1987) inTeratocarcinomas and Embryonic Stem Cells: a Practical Approach (E. J.Robertson, ed.) Oxford: IRL Press, p. 71-112). The linearized CMDtargeting vector was electroporated into AB-1 cells by the methodsdescribed Hasty et al. (Hasty, P. R. et al. (1991) Nature 350: 243-246).Electroporated cells were plated into 100 mm dishes at a density of1-2×106 cells/dish. After 24 hours, G418 (200 micrograms/ml of activecomponent) and FIAU (5×10-7 M) were added to the medium, anddrug-resistant clones were allowed to develop over 8-9 days. Clones werepicked, trypsinized, divided into two portions, and further expanded.Half of the cells derived from each clone were then frozen and the otherhalf analyzed for homologous recombination between vector and targetsequences.

DNA analysis was carried out by Southern blot hybridization. DNA wasisolated from the clones as described Laird et al. (Laird, P. W. et al.,(1991) Nucleic Acids Res. 19: 4293). Isolated genomic DNA was digestedwith SpeI and probed with a 915 by SacI fragment, probe A (FIG. 1),which hybridizes to a sequence between the mu intronic enhancer and themu switch region. Probe A detects a 9.9 kb SpeI fragment from the wildtype locus, and a diagnostic 7.6 kb band from a mu locus which hashomologously recombined with the CMD targeting vector (the neoexpression cassette contains a SpeI site). Of 1132 G418 and FIAUresistant clones screened by Southern blot analysis, 3 displayed the 7.6kb Spe I band indicative of homologous recombination at the mu locus.These 3 clones were further digested with the enzymes BglI, BstXI, andEcoRI to verify that the vector integrated homologously into the mugene. When hybridized with probe A, Southern blots of wild type DNAdigested with BglI, BstXI, or EcoRI produce fragments of 15.7, 7.3, and12.5 kb, respectively, whereas the presence of a targeted mu allele isindicated by fragments of 7.7, 6.6, and 14.3 kb, respectively. All 3positive clones detected by the SpeI digest showed the expected BglI,BstXI, and EcoRI restriction fragments diagnostic of insertion of theneo cassette into the Cmul exon.

Generation of Mice Bearing the Mutated Mu Gene.

The three targeted ES clones, designated number 264, 272, and 408, werethawed and injected into C57BL/6J blastocysts as described by Bradley(Bradley, A. (1987) in Teratocarcinomas and Embryonic Stem Cells: aPractical Approach. (E. J. Robertson, ed.) Oxford: IRL Press, p.113-151). Injected blastocysts were transferred into the uteri ofpseudopregnant females to generate chimeric mice representing a mixtureof cells derived from the input ES cells and the host blastocyst. Theextent of ES cell contribution to the chimera can be visually estimatedby the amount of agouti coat coloration, derived from the ES cell line,on the black C57BL/6J background. Clones 272 and 408 produced only lowpercentage chimeras (i.e. low percentage of agouti pigmentation) butclone 264 produced high percentage male chimeras. These chimeras werebred with C57BL/6J females and agouti offspring were generated,indicative of germline transmission of the ES cell genome. Screening forthe targeted mu gene was carried out by Southern blot analysis of BOdigested DNA from tail biopsies (as described above for analysis of EScell DNA). Approximately 50% of the agouti offspring showed ahybridizing BglI band of 7.7 kb in addition to the wild type band of15.7 kb, demonstrating a germline transmission of the targeted mu gene.

Analysis of Transgenic Mice for Functional Inactivation of Mu Gene.

To determine whether the insertion of the neo cassette into CmuI hasinactivated the Ig heavy chain gene, a clone 264 chimera was bred with amouse homozygous for the JHD mutation, which inactivates heavy chainexpression as a result of deletion of the JH gene segments (Chen et al.,(1993) Immunol. 5: 647-656). Four agouti offspring were generated. Serumwas obtained from these animals at the age of 1 month and assayed byELISA for the presence of murine IgM. Two of the four offspring werecompletely lacking IgM (Table 1). Genotyping of the four animals bySouthern blot analysis of DNA from tail biopsies by BglI digestion andhybridization with probe A (FIG. 1), and by StuI digestion andhybridization with a 475 by EcoRI/StuI fragment (ibid.) demonstratedthat the animals which fail to express serum IgM are those in which oneallele of the heavy chain locus carries the JHD mutation, the otherallele the Cmu1 mutation. Mice heterozygous for the JHD mutation displaywild type levels of serum Ig. These data demonstrate that the Cmu1mutation inactivates expression of the mu gene.

TABLE 1 Level of serum IgM, detected by ELISA, for mice carrying boththe CMD and JHD mutations (CMD/JHD), for mice heterozygous for the JHDmutation (+/JHD), for wild type (129Sv × C57BL/6J)F1 mice (+/+), and forB cell deficient mice homozygous for the JHD mutation (JHD/JHD). SerumIgM Mouse (micrograms/ml) Ig H chain genotype 42 <0.002 CMD/JHD 43 196+/JHD 44 <0.002 CMD/JHD 45 174 +/JHD 129 × BL6 F1 153 +/+ JHD <0.002JHD/JHD

Example 2 Generation of HCo12 Transgenic Mice

The HCo12 Human Heavy Chain Transgene.

The HCo12 transgene was generated by coinjection of the 80 kb insert ofpHC2 (Taylor et al., 1994, Int. Immunol., 6: 579-591) and the 25 kbinsert of pVx6. The plasmid pVx6 was constructed as described below.

An 8.5 kb HindIII/SalI DNA fragment, comprising the germline humanVH1-18 (DP-14) gene together with approximately 2.5 kb of 5′ flanking,and 5 kb of 3′ flanking genomic sequence was subcloned into the plasmidvector pSP72 (Promega, Madison, Wis.) to generate the plasmid p343.7.16.A 7 kb BanaHI/HindIII DNA fragment, comprising the germline human VH5-51(DP-73) gene together with approximately 5 kb of 5′ flanking and 1 kb of3′ flanking genomic sequence, was cloned into the pBR322 based plasmidcloning vector pGPlf (Taylor et al. 1992, Nucleic Acids Res. 20:6287-6295), to generate the plasmid p251f. A new cloning vector derivedfrom pGP1f, pGP1k (Seq. ID #1), was digested with EcoRV/BamHI, andligated to a 10 kb EcoRV/BamHI DNA fragment, comprising the germlinehuman VH3-23 (DP47) gene together with approximately 4 kb of 5′ flankingand 5 kb of 3′ flanking genomic sequence. The resulting plasmid,p112.2RR.7, was digested with BamHI/SalI and ligated with the 7 kbpurified BamHI/SalI insert of p251 f. The resulting plasmid, pVx4, wasdigested with XhoI and ligated with the 8.5 kb XhoI/SalI insert ofp343.7.16. A clone was obtained with the VH1-18 gene in the sameorientation as the other two V genes. This clone, designated pVx6, wasthen digested with NotI and the purified 26 kb insertcoinjected—together with the purified 80 kb NotI insert of pHC2 at a 1:1molar ratio—into the pronuclei of one-half day (C57BL/6J×DBA/2J)F2embryos as described by Hogan et al. (B. Hogan et al.,, Manipulating theMouse Embryo, A Laboratory Manual, 2nd edition, 1994, Cold Spring HarborLaboratory Press, Plainview N.Y.). Three independent lines of transgenicmice comprising sequences from both Vx6 and HC2 were established frommice that developed from the injected embryos. These lines aredesignated (HCo12)14881, (HCo12)15083, and (HCo12)15087. Each of thethree lines were then bred with mice comprising the CMD mutationdescribed in Example 1, the JKD mutation (Chen et al,. 1993, EMBO J. 12:811-820), and the (KCo5)9272 transgene (Fishwild et al. 1996, NatureBiotechnology 14: 845-851). The resulting mice express human heavy andkappa light chain transgenes in a background homozygous for disruptionof the endogenous mouse heavy and kappa light chain loci.

Example 3 Generation of Human IgG Kappa Anti-Human CTLA-4 MonoclonalAntibodies

Cell Based Antigen

A DNA segment encoding a fusion protein comprising sequences from thehuman CTLA-4 and the murine CD3zeta genes was constructed by PCRamplification of cDNA clones together with bridging syntheticoligonucleotides. The encoded fusion protein contains the followingsequences: i. human CTLA-4 encoding amino acids 1-190 (containing thesignal peptide, the extracellular domain of human CTLA-4 and theentirety of the presumed transmembrane sequence of human CTLA-4) and ii.murine CD3zeta from amino acid 52 to the carboxy terminus (Weissman etal. (1988) Science 239: 1018-1021). The amplified PCR product was clonedinto a plasmid vector and the DNA sequence was determined. The clonedinsert was then subcloned into the vector pBABE (which contains a geneencoding for puromycin resistance (Morganstern, J P and Land, H Nucl.Acids Res. 18: 3587-96 (1990)) to create pBABE-huCTLA-4/CD3z.pBABE-huCTLA-4/CD3z was transfected into the retroviral packaging line,ψ-2, and a pool of puromycin resistant cells were selected. These cellswere co-cultured with the murine T cell hybridoma BW5147 (ATCC #TIB-47).After 2 days of co-culture the non-adherent BW5147 cells were removedand selected for resistance to puromycin. The puromycin resistant cellpool was subcloned by limiting dilution and tested for surfaceexpression of human CTLA-4 by FACS. A clone expressing high levels ofhuman CTLA-4 at the cell surface was selected.

Soluble Antigen

Recombinant CTLA-4 fusion protein comprising the extracellular domain ofhuman CTLA-4 was purchased from R&D Systems (Cat. #325-CT-200).Extracellular CTLA-4 fragment was prepared by proteolytic cleavage ofthe CTLA-4 fusion protein at a Factor Xa protease cleavage site locatedafter the C-terminus of the CTLA-4 extracellular domain. Fusion proteinwas treated with Factor Xa at a ratio of 50:1 of fusion protein toFactor Xa, and the CTLA-4 fragment was isolated by passage over proteinG-Sepharose and Mono Q HPLC. Fractions were tested for the presence ofhuman CTLA-4 dimer were by SDS-PAGE and by binding to cells expressingmouse 137 molecules (LtkmB7.1: mouse Ltk(−) cells transfected with amouse B7.1 cDNA clone expression vector). Positive fractions were pooledand dialyzed into PBS buffer.

Transgenic Mice

Two different strains of mice were used to generate CTLA-4 reactivemonoclonal antibodies. Strain ((CMD)++; (JKD)++; (HCo7)11952+/++;(KCo5)9272+/++), and strain ((CMD)++; (JKD)++; (HCo12)15087+/++;(KCo5)9272+/++). Each of these strains are homozygous for disruptions ofthe endogenous heavy chain (CMD) and kappa light chain (JKD) loci. Bothstrains also comprise a human kappa light chain transgene (KCo5), withindividual animals either hemizygous or homozygous for insertion #11952.The two strains differ in the human heavy chain transgene used. Micewere hemizygous or homozygous for either the HCo7 or the HCo12transgene. The CMD mutation is described above in Example 1. Thegeneration of (HCo12)15087 mice is described in Example 2. The JKDmutation (Chen et al. 1993, EMBO J. 12: 811-820) and the (KCo5)9272(Fishwild et al. 1996, Nature Biotechnology 14: 845-851) and (HCo7)11952mice, are described in U.S. Pat. No. 5,770,429 (Lonberg & Kay, Jun. 23,1998).

Immunization

Transgenic mice were initially immunized i.p. with 1-3×10⁷ cellsin PBS,or with 10-50 ug soluble fusion protein in adjuvant (either completeFreund's or Ribi). Immunized mice were subsequently boosted every 2 to 4weeks i.p. with 1-3×10⁷ cells in PBS. Animals were kept on protocol for2 to 5 months. Prior to fusion, animals were boosted i.v. on days −3 and−2 with approximately 10⁶ cells, or with 10-20 ug soluble antigen(fusion protein or fusion protein and extracellular fragment). Someanimals also received fusion protein i.v. on day −4. Successful fusionsresulting in CTLA-4 reactive IgG kappa monoclonal antibodies wereobtained from mice immunized by a variety of different protocols,including cells only, soluble antigen only, and cell immunizationsfollowed by soluble antigen given i.v. prior to fusion.

Fusions

Spleen cells were fused to mouse myeloma cells (line P3 X63 Ag8.6.53,ATCC CRL 1580, or SP2/0-Ag14, ATCC CRL 1581) by standard procedures(Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor N.Y.; Kennett et al. 1980,Monoclonal Antibodies, Hybridomas: A New Dimension in BiologicalAnalysis. Plenum, New York; Oi and Hertzenberg, 1980, ImmunoglobulinProducing Hybrid Cell Lines, in Selected Methods In Cellular Immunology,ed. Mishell and Shiigi, pp. 357-372. Freeman, San Francisco; Halk, 1984,Methods in Enzymology: Plant Molecular Biology, ed. Weissbach andWeissbach, pp. 766-780, Academic Press, Orlando, Fla.). Cells werecultured in DMEM, 10% FBS, OPI (Sigma O-5003), BME (Gibco 21985-023), 3%Origen Hybridoma Cloning Factor (Igen 1050-0615), and 5% P388d1 (ATCCTIB 63) conditioned media. HAT or HT supplement was added to the mediumduring initial growth and selection.

Hybridoma Screening

To identify hybridomas secreting human IgG kappa antibodies, ELISAplates (Nunc MaxiSorp) were coated overnight at 4° C. with 100 μl/wellgoat anti-human Fcgamma specific antibody (Jackson Immuno Research#109-006-098) at 1 ug/ml in PBS. Plates were washed and blocked with 100ul/well PBS-Tween containing 1% BSA. Fifty ul cell culture supernatantwas added followed by a 1-2 hour incubation. Plates were washed and thenincubated for one hour with 100 ul/well goat anti-Kappa light chainconjugated to alkaline phosphatase or horseradish peroxidase (Sigma#A-3813, or #A-7164). Plates were washed three times in PBS-Tweenbetween each step. An analogous assay was used to identify hybridomasthat secrete human antibodies reactive with human CTLA-4. This assay wasidentical except that the ELISA plates were coated with recombinantCTLA-4 fusion protein instead of goat anti-human Fcgamma antibody.

Characterization of Monoclonal Antibodies

Seventy two hybridomas that were shown by ELISA to secrete human IgGkappa binding to human CTLA-4 were subcloned. Forty seven of thesesubclones were tested to determine if the secreted human antibodies bindto CTLA-4 expressing cells, and if the antibodies inhibit soluble CTLA-4from binding to cells expressing B7. Binding was determined by flowcytometry. To measure inhibition, 50 microliters of each supernatant wasincubated with 10⁵ LtkmB7.1 cells and 25 ng recombinant CTLA-4 fusionprotein. Mean channel fluorescence was then determined by flowcytometry. FIG. 2 shows inhibition of soluble CTLA-4 binding to cellsexpressing B7.1. Mean channel fluorescence (MCF) of LtkmB7.1 cellsstained with recombinant human CTLA-4 fusion protein was determined inthe presence of hybridoma supernatant. Hybridomas that secrete blockingantibodies resulted in lower MCF values. BNI3.1 (Cat.#34580D,Pharmingen, San Diego, Calif.) was used as a positive control mousemonoclonal antibody that blocks CTLA-4/B7 binding.

Approximately 40% of the hybridomas appear to strongly inhibit CTLA-4binding to the B7 ligand.

Antibodies from clones 10D1.3, 4B6.12, and 11E8, were then assayed byBIAcore (Biacore AB, Uppsala, Sweden) to determine binding kinetics.Purified recombinant CTLA-4 extracellular fragment was coupled to theCM5 sensor chip @ 1200 RU. Binding was measured by adding antibody atconcentrations of 0.25, 0.5, 1, 2.5, and 5 ug/ml at a flow rate of 5μl/min. The binding curves were fit to a Langmuir binding model usingBIAevaluation software (Biacore AB, Uppsala, Sweden). Antibodies werepurified by protein-A Sepharose chromatography. Determined on and offrates are shown in Table 2:

TABLE 2 Kinetics of binding of human IgG kappa antibodies to recombinantCTLA-4 immobilized on a surface. Hybridoma ka (1/Ms) kd (1/s) Ka (1/M)10D1.3 4.1 × 10⁵ 1.0 × 10⁻⁴ 4 × 10⁹ 4B6.12 5.1 × 10⁵ 1.3 × 10⁻⁴ 4 × 10⁹11E8 4.3 × 10⁵ 1.8 × 10⁻⁴ 2 × 10⁹

Serial dilutions of 10 different human IgG kappa anti-human CTLA-4monoclonal antibodies (3A4, 9A5, 2E2, 2E7, 4B6, 4E10, 5C4, 5G1, 11E8,and 11G1) were added to microtiter wells coated with recombinant CTLA-4fusion protein. After a 2 hour incubation, biotinylated antibody 11E8was added to each well at a concentration of 0.1 ug/ml. The samples wereincubated for 30 minutes, washed, and bound antibody detected withalkaline phosphatase/streptavidin conjugate. The titrations are shown inFIG. 3. Antibody 11E8 binding was blocked by itself and 7 of the otherhuman antibodies. However, binding was not blocked by antibodies 3A4 or9A5. Reciprocal binding experiments showed that 11E8 binding did notblock either 3A4 or 9A5 binding to CTLA-4.

DNA Sequence

RNA was extracted from approximately 2×10⁶ cells of each subclonedhybridoma cell line and used to synthesize cDNA using reagents andprotocols from Invitrogen (Micro-FastTrack and cDNA Cycle: Cat.#L1310-01, and #K1520-02, Invitrogen, Carlsbad, Calif.). Humanimmunoglobulin heavy and kappa light chain V region fragments wereamplified by PCR using pfu polymerase (Stratagene, La Jolla, Calif.),degenerate FR1 primers and unique constant region primers. The resultingPCR fragments were cloned into the pCR-Blunt vector (Invitrogen,Carlsbad, Calif.) and the sequence of the insert determined. Thepreliminary sequences for the heavy and light chain fragment ofhybridoma 10D1.3 are shown in FIG. 4. The determined sequences for theheavy and light chain fragment of hybridoma 10D1.3 are shown in FIG. 5through FIG. 8.

TABLE 3 CDR sequences of light and heavy chains for MAbs10D1, 4B6,and 1E2. SEQ ID SEQ ID SEQ ID Chain HuMAb CDR1 NO: CDR2 NO:CDR3 NO: Light 10D1 RASQSVGSSYLA 24 GAFSRAT 29 QQYGSSPWT 35 Chain 4B6RASQSVGSSFLA 25 GASSRAT 30 QQYGSSPWT 35 1E2 RASQGISSWLA 26 AASSLQS 31QQYNSYPPT 36 Heavy 10D1 SYTMH 27 FISYDGNNKYYADSVKG 32 TGWLGPFDY 37 Chain4B6 SYTMH 27 FISYDGSNKHYADSVKG 33 TGWLGPFDY 37 1E2 SYGMH 28VIWYDGSNKYYADSVKG 34 APNYIGAFDV 38

Example 4 Use of Partial Antibody Sequences to Express Intact Antibodies

Antibodies interact with target antigens predominantly through aminoacid residues that are located in the six heavy and light chaincomplimentarily determining regions (CDR's). For this reason, the aminoacid sequences within CDR's are more diverse between individualantibodies than sequences outside of CDR's. Because CDR sequences areresponsible for most antibody-antigen interactions, it is possible toexpress recombinant antibodies that mimic the properties of specificnaturally occurring antibodies by constructing expression vectors thatinclude CDR sequences from the specific naturally occurring antibodygrafted onto framework sequences from a different antibody withdifferent properties (Jones et al. 1986, Nature 321, 522-525). Suchframework sequences can be obtained from public DNA databases thatinclude germline antibody gene sequences. These germline sequences willdiffer from mature antibody gene sequences because they will not includecompletely assembled variable genes, which are formed by V(D)J joiningduring B cell maturation. Germline gene sequences will also differ fromthe sequence of a high affinity secondary repertoire antibody atindividual nucleotides because of somatic mutations. However, somaticmutations are not distributed evenly across the variable region. Forexample, somatic mutations are relatively infrequent in theamino-terminal portion of framework region 1 and in the carboxy-terminalportion of framework region 4. Furthermore, many somatic mutations donot significantly alter the binding properties of the antibody. For thisreason, it is not necessary to obtain the entire DNA sequence of aparticular antibody in order to recreate an intact recombinant antibodyhaving binding properties similar to those of the original antibody (seePCT/US99/05535 filed on Mar. 12, 1999, which is herein incorporated byreference for all purposes). Partial heavy and light chain sequencespanning the CDR regions is typically sufficient for this purpose. Thepartial sequence is used to determine which germline variable andjoining gene segments contributed to the recombined antibody variablegenes. The germline sequence is then used to fill in missing portions ofthe variable region. Heavy and light chain leader sequences are cleavedduring protein maturation and do not contribute to the properties of thefinal antibody. For this reason it is not necessary to use thecorresponding germline leader sequence for expression constructs. To addmissing sequences, cloned cDNA sequences can be combined with syntheticoligonucleotides by ligation or PCR amplification. Alternatively, theentire variable region can be synthesized as a set of short,overlapping, oligonucleotides and combined by PCR amplification tocreate an entirely synthetic variable region clone. This process hascertain advantages such as elimination or inclusion of particularrestriction sites, or optimization of particular codons.

The nucleotide sequences of heavy and light chain transcripts from ahybridomas are used to design an overlapping set of syntheticoligonucleotides to create synthetic V sequences with identical aminoacid coding capacities as the natural sequences. The synthetic heavy andkappa light chain sequences can differ from the natural sequences inthree ways: strings of repeated nucleotide bases are interrupted tofacilitate oligonucleotide synthesis and PCR amplification; optimaltranslation initiation sites are incorporated according to Kozak's rules(Kozak, 1991, J. Biol. Chem. 266, 19867-19870); and, HindIII sites areengineered upstream of the translation initiation sites.

For both the heavy and light chain variable regions, the optimizedcoding, and corresponding non-coding, strand sequences are broken downinto 30-50 nucleotide segments such that the breaks between nucleotidesfor the coding strand sequence occur at approximately the midpoint ofthe corresponding non-coding oligonucleotide. Thus, for each chain, theoligonucleotides can be assemble into overlapping double stranded setsthat completely span the desired sequence. These oligonucleotides arecombined into pools that span segments of 150-400 nucleotides. The poolsare then used as templates to produce PCR amplification products of150-400 nucleotides. Typically, a single variable region oligonucleotideset will be broken down into two pools which are separately amplified togenerate two overlapping PCR products. These overlapping products arethen combined by PCR amplification to form the complete variable region.It may also be desirable to include an overlapping fragment of the heavyor light chain constant region (including the BbsI site of the kappalight chain, or the AgeI site if the gamma heavy chain) in the PCRamplification to generate fragments that can easily be cloned into theexpression vector constructs.

The reconstructed heavy and light chain variable regions are thencombined with cloned promoter, translation initiation, constant region,3′ untranslated, polyadenylation, and transcription termination,sequences to form expression vector constructs. The heavy and lightchain expression constructs can be combined into a single vector,co-transfected, serially transfected, or separately transfected intohost cells which are then fused to form a host cell expressing bothchains.

Plasmids for use in construction of expression vectors for human IgGkare described below. The plasmids were constructed so that PCR amplifiedV heavy and V kappa light chain cDNA sequences could be used toreconstruct complete heavy and light chain minigenes. These plasmids canbe used to express completely human, or chimeric IgG1k or IgG4kantibodies, Similar plasmids can be constructed for expression of otherheavy chain isotypes, or for expression of antibodies comprising lambdalight chains.

The kappa light chain plasmid, pCK7-96 (SEQ ID NO:39), includes thekappa constant region and polyadenylation site, such that kappasequences amplified with 5′ primers that include HindIII sites upstreamof the initiator methionine can be digested with HindIII and BbsI, andcloned into pCK7-96 digested with HindIII and BbsI to reconstruct acomplete light chain coding sequence together with a polyadenylationsite. This cassette can be isolated as a HindIII/NotI fragment andligated to transcription promoter sequences to create a functionalminigene for transfection into cells.

The gamma1 heavy chain plasmid, pCG7-96 (SEQ ID NO:40), includes thehuman gamma1 constant region and polyadenylation site, such that gammasequences amplified with 5′ primers that include HindIII sites upstreamof the initiator methionine can be digested with HindIII and AgeI, andcloned into pCG7-96 digested with HindIII and AgeI to reconstruct acomplete gamma1 heavy chain coding sequence together with apolyadenylation site. This cassette can be isolated as a HindIII/SalIfragment and ligated to transcription promoter sequences to create afunctional minigene for transfection into cells.

The gamma4 heavy chain plasmid, pG4HE (SEQ ID NO:41), includes the humangamma4 constant region and polyadenylation site, such that gammasequences amplified with 5′ primers that include HindIII sites upstreamof the initiator methionine can be digested with HindIII and AgeI, andcloned into pG4HE digested with HindIII and AgeI to reconstruct acomplete gamma4 heavy chain coding sequence together with apolyadenylation site. This cassette can be isolated as a HindIII/EcoRIfragment and ligated to transcription promoter sequences to create afunctional minigene for transfection into cells.

A number of different promoters (including but not limited to CMV,ubiquitin, SRalpha, and beta-actin) can be used to express thereconstructed heavy and light chain genes. For example the vectorpcDNA3.1+ (Invitrogen, Carlsbad, Calif.), can be cleaved with Hindu andeither NotI, XhoI, or EcoRI, for ligation with either the kappa, gamma1,or gamma4 cassettes described above, to form expression vectors that canbe directly transfected into mammalian cells.

Example 5 10D.1 Binding to CTLA-4

A. 10D1 Binding to Purified Recombinant Human CTLA-4

Binding of 10D1 to purified recombinant human CTLA-4 was shown by ELISAusing standard methods and procedures (FIG. 9 and FIG. 10). Microtiterplates coated with purified CTLA-4 were incubated with varyingconcentration of 10D1, and then developed with goat anti-human IgGF(ab′)₂ conjugated to alkaline phosphatase. The data demonstratedose-dependent binding of 10D1 that is well fit to a 4-parameter curve(correlation coefficient is −1.0). The half-maximal binding at 15 ng/mlreflects the high binding capacity of 10D1 to CTLA-4. Saturation ofbinding was observed at approximately 0.1 μg/ml.

B. 10D.1 Binding to CTLA-4 Expressed on the Plasma Membrane of T-Cells

In order to demonstrate binding of 10D1 to CTLA-4 expressed on theplasma membrane of T-cells, the results in FIG. 10 from a flowcytometric assay are shown. The flow cytometric assay was used with aT-cell line transfected to express high levels of human CTLA-4(designated 58αβCTLA-4/CD3zeta cells). Varying concentrations offluoresceinated 10D1 (10D1-FITC) were incubated with 58αβCTLA-4 cells.The cell associated fluorescence was determined by flow cytometry. Asseen with the purified CTLA4, 10D1 bound to CTLA4-expressing cells in adose-dependent manner that was well fit to a 4-paramater equation(correlation coefficient is −0.999). The half-maximal binding was 190ng/ml, and saturation was achieved at 2 μg/ml. 10D1 did not bind to anyCTLA4-negative cell lines tested, including SKBR-3, BT474 and MCF10Abreast epithelial tumors and L540 Hodgkin's tumor cells, nor did it bindto cells expressing murine CTLA-4. These data indicate the specificityof 10D1 for human CTLA. However, 10D1 was shown to cross-react withmacaque CTLA-4 (see below).

C. Cross-Reactivity of 10D1 with Normal Human Tissues

In this study, a fluoresceinated form of the test article (10D1-FITC)was used to evaluate binding. The objective of the study was to evaluatepotential cross-reactivity of 10D1-FITC with cryosections of normalhuman tissues. No unanticipated cross-reactivity was observed.

The study was conducted in accordance with the Food and DrugAdministration's Good Laboratory Practice (GLP) Regulations (21 CFR Part58). The human tissue panel included all the tissue on the “suggestedlist of human tissues to be used for immunohistochemical investigationsof cross reactivity’ in Annex II of the EC CPMP Guideline III/5271/94,“Production and quality control of monoclonal antibodies” and all thetissues recommended in the 1997 US FDA/CBER “Points to Consider in theManufacture and Testing of Monclonal Antibody Products for Human Use”.

Using an indirect immunoperoxidase method, 10D1-FITC specificallystained positive control, human CTLA4-expressing, 58αβCTLA4CD3zeta cellsas well as positive control lymphocytes in human tonsil. 10D1-FITCreactivity was moderate to intense and two concentrations of antibodywere examined (10 μg/ml and 2.5 μg/ml). In both positive control58αβCTLA4CD3zeta and positive control human tonsillar lymphocytes,10D1-FITC specifically stained discrete, round, granules at membrane andin the cytoplasm immediately below the membrane. Reactivity was observedwith occasional follicular, interfollicular, and subepitheliallymphocytes. Less than 1-2% of all tonsillar lymphocytes were reactivewith 10D1-FITC.

10D1-FITC did not react with negative control human brain (cerebellum).An isotype-matched negative control antibody (HulgG1-k-FITC) did notspecifically bind to either the positive control human CTLA4-expressing58αβCTLA4CD3zeta or human tonsil; nor did it bind specifically tonegative control human brain (cerebellum).

To determine cross-reactivity, 10D1-FITC was applied to a panel ofnormal human tissues at two concentrations (10 μg/ml and 2.5 μg/ml).Specific 10D1-FITC reactivity was observed for lymphocytes in the tonsil(3/3 donors), submucosal lymphoid nodule in the colon (gastrointestinaltract-colon [1/3 donors]), and blood smears (2/3 donors).

Immunoreactive cells were identified as lymphocytes based on typicalmorphology (round molecular cells with large nucleus:cytoplasm ratio andscant cytoplasm, lack of dendritic processes, 10-15 μm in diameter) andlocation within the tissues (e.g., typical location within lymphoidtissues). In the tonsils from all three donors (test tissues),lymphocytes, 10D 1-FITC specifically stained discrete, round, granulesat membrane and in the cytoplasm immediately below the membrane.Reactivity was observed with occasional follicular, interfollicular andsubepithelial lymphocytes. Less than 1-2% of all tonsillar lymphcyteswere reactive with 10D1-FITC.

In 1/3 donors examined, 10D1-FITC also specifically stained discretegranules in occasional follicular and interfollicular lymphocyteslocated in submucosal lymphoid nodules in the colon (gastrointestinaltract-colon [large intestine]). Again, discrete membrane granules werestained.

In peripheral blood smears from two of the three donors examined,10D1-FITC specifically stained discrete granules approximately 1 μm indiameter associated with the membrane of rare lymphocytes. The granuleswere arranged in a ring or in a curved pattern. Less than 1-2% of allperipheral blood leukocytes were reactive with 10D1-FITC.

TABLE 4 Cross-Reactivity of MAb 10D1 With Normal Human Tissues NegativeControl Antibody Test Article HulgG1-k- 10D1-FITC FITC Assay Tissue 10μg/ml 2.5 μg/ml 10 μg/ml 2.5 μg/ml Control * β₂-microglobulin PositiveControl 3-4+ 2-4+ Neg Neg Neg Pos 58αβCTLA4CD3zeta cells PositiveControl Lymphocytes 2-3+ 2-3+ Neg Neg Neg Pos in human tonsil NegativeControl Human brain - Neg Neg Neg Neg Neg Pos cerebellum Adrenal Neg NegNeg Neg Neg Pos Blood Pos Neutrophils Neg Neg Neg Neg Neg PosLymphocytes 2+ Neg Neg Neg Neg Pos (rare) Eosinophils Neg Neg Neg NegNeg Pos Monocytes Neg Neg Neg Neg Neg Pos Platelets Neg Neg Neg Neg NegPos Blood Vessel (endothelium) Detailed under individual tissuesExamined in all tissues Bone Marrow Neg Nag Neg Neg Neg Pos Brain -Cerebellum Neg Neg Neg Neg Neg Pos Brain - Cerebrum (cortex) Neg Neg NegNeg Neg Pos Breast (mammary gland) Neg Neg Neg Neg Neg Pos Eye Neg NegNeg Neg Neg Pos Gastrointestinal Tract - Colon 2-3+ 2-3+ Neg Neg Neg Pos(large intestine) Submucosal lymphoid nodule (occasional follicular andinterfollicular lymphocytes) Gastrointestinal Tract - Colon Neg Neg NegNeg Neg Pos (large intestine) Other elements Gastrointestinal Tract -Neg Neg Neg Neg Neg Pos Esophagus Gastrointestinal Tract - Small Neg NegNeg Neg Neg Pos intestine Gastrointestinal Tract - Neg Neg Neg Neg NegPos Stomach Heart Neg Neg Neg Neg Neg Pos Kidney (glomerulus, tubule)Neg Neg Neg Neg Neg Pos Liver Neg Neg Neg Neg Neg Pos Lung Neg Neg NegNeg Neg Pos Lymph Node Neg Neg Neg Neg Neg Pos Ovary Neg Neg Neg Neg NegPas Fallopian Tube (oviduct) Neg Neg Neg Neg Neg Pos Pancreas Neg NegNeg Neg Neg Pos Parathyroid Nag Neg Neg Neg Neg Pos Peripheral Nerve NegNeg Neg Neg Neg Pos Pituitary Neg Neg Neg Neg Neg Pos Placenta Neg NegNeg Neg Neg Pos Prostate Neg Neg Neg Neg Neg Pos Salivary Gland Neg NegNeg Neg Neg Pos Skin Neg Neg Neg Neg Neg Pos Spinal Cord Neg Neg Neg NegNeg Pos Spleen Neg Neg Neg Neg Neg Pos Striated (Skeletal) Muscle NegNeg Neg Neg Neg Pos Testis Neg Neg Neg Neg Neg Pos Thymus Neg Neg NegNeg Neg Pos Thyroid Neg Neg Neg Neg Neg Pos Tonsil Lymphocytes 2+ 1-2+Neg Neg Neg Pos (occasional follicular, interfollicular andsubepithelial lymphocytes) Tonsil Other elements Neg Neg Neg Neg Neg PosUreter Neg Neg Neg Neg Neg Pos Urinary Bladder Neg Neg Neg Neg Neg PosUterus - Body (endometrium) Neg Nei Neg Neg Neg Pos Uterus - Cervix NegNeg Neg Neg Neg Pos * omission of test antibody

D. Specific Reactivity of 10D.1 with Macaque CTLA-4

Specific reactivity with macaque CTLA-4 was demonstrated using T-cellstransfected to express the macaque CTLA-4 at high levels (Table 5).These data suggest that the CTLA-4 epitope for 10D1 is conserved betweenmacaque and humans, therefore macaque is a good model to evaluate invivo safety of anti-CTLA4 HuMAb 10D1.

TABLE 5 reactivity of isotype reactivity of Species control (MFI) 10D1(MFI) human CTLA4 3 662 macaque CTLA4 4 606 murine CTLA4 5 5 (negativecontrol)

MAb 10D1 (10 μg/ml) was incubated with cell lines expressing recombinantCTLA-4 from various species, and detected by FITC-anti human IgG. Thecell-associated fluorescence was determined by FACScan and reported asmean fluorescence intensity (MFI). These data show that MAb 10D1 reactswell with macaque and human CTLA-4, but not with murine CTLA-4.

Example 6 10D1 Blocking of CTLA-4 to B7 Ligands

In order to show that 10D1 binding to CTLA-4 blocks the interaction ofCTLA-4 with CTLA-4 ligands, B7.1 and B7.2, competition assays wereperformed by flow cytometry (FIG. 11 and FIG. 12). As shown in FIG. 11,FITC-labeled human B7.2-Ig fusion protein was incubated with 58αβCTLA4T-cells and various concentrations of 10D1 MAb. In FIG. 12, FITC-labeledCTLA4-Ig fusion protein was incubated with murine B7.1 transfected cellsand various concentrations of 10D1 MAb.

The competition assays demonstrate the ability of 10D1 to efficientlyinhibit CTLA4-B7 interactions at low concentrations (1-10 μg/ml). Theeffective concentration would likely be much lower under physiologicalconditions, which would have far lower concentrations of CTLA-4 and B7molecules. Similar data was obtained using biotinylated reagents inELISA assays.

These in vitro studies demonstrate that MAb 10D1 binds human CTLA-4 withhigh affinity and specificity and that binding of 10D1 abrogatesinteraction between B7 costimulatory molecules and CTLA-4. These datafor 10D1 are consistent with the in vitro activity profiles foranti-murine CTLA-4 antibodies that have demonstrated efficacy in murinetumor models.

Example 7 Epitope Mapping of 10D.1

Competitive ELISAs were done with biotin labeled and unlabeledantibodies to determine CTLA-4 epitope specificity. Four anti-CTLA-4epitope binding groups were identified among the human antibodies, andan additional two epitopes were defined by the commercial murinemonclonal antibodies BNI3 (Pharmingen, San Diego, Calif.), and 8H5(Ancell Corp. Bayport, Mn). FIGS. 3, and 13A-13G show results ofcompetitive binding assays that demonstrate differential competitionamong the antibodies for binding to CTLA-4. These results are summarizedin Table 6.

Antibodies in anti-CTLA-4 epitope binding groups 4a and 4b have similarbinding characteristics, and additionally are strong blockers ofCTLA-4-Ig binding to cell surface expressed B7.1 (Table 6). For example,FIG. 3 shows results with biotin labeled 11E8 antibody and 10 unlabeledantibodies (3A4, 9A5, 2E2, 2E7, 4B6, 4E10, 5C4, 5G1, 11E8 and 11G1).Antibody 11E8 binding was blocked by itself and 7 of the other humanantibodies in epitope groups 4a and 4b. However, binding of 11E8 was notblocked by antibodies 3A4 or 9A5(epitope groups 1 and 2). Reciprocalbinding experiments showed that 11E8 binding did not block either 9A5 or3A4 binding to CTLA-4 (FIGS. 13A and 13B). Similar results are shown forepitope group 4a antibodies 10D1 and murine antibody 147 (FIGS. 13D and13F). Antibodies in epitope group 4b (FIG. 13E) are similar to group 4aantibodies with the exception that the epitope 4b antibodies competewith epitope group 2 antibodies in reciprocal binding experiments (FIG.13B). Human antibodies that belong to epitope groups 3, 4a and 4b areeffective blockers of CTLA-4/B7.1 binding (FIG. 3, and Table 6).

TABLE 6 CTLA-4 MABs: Epitope and CTLA-4/B7.1 Blocking Properties Blocksbinding Monoclonal of CTLA-4-Ig to Epitope Antibody Competition forCTLA-4 Binding B7.1 on Ltk mB7.1 1 9A5 No competition from groups 3, 4a,4b, 5, and 6 No Weak Competition form group 2 2 3A4 One way competitionfrom groups 1, 4b, 5 and 6 No 1E2 No competition with 4a. Weakcompetition form group 3 3 5A8 Competes with 4a and 4b. Some competitionwith 2. Yes No competition form 1 and 5 4a 10D1 Cross competes with allmembers of 4b. Yes 147* Competition from 6 (non-reciprocal) 11E8 Nocompetition with 1, 2, and 5. 11G1 Weak competition with 3. 4E10 5C43F10 4b 4B6 Cross competes with all members of 4a Yes 4A1 Competes with2 2E2 Weak competition with 3. 2E7 No competition with 1, and 5. 2G1Competition from 6 (non-reciprocal) 5 BNI3** Competes with 6, nocompetition with groups 1 to 4 Yes 6 8H5*** Competes with 5, nocompetition with groups 1 to 4 Yes Competition with group 3 not tested*Murine monoclonal antibody **Available from Pharmingen, BNI3 Catalog #34580 D, San Diego CA. ***Available from Ancell, ANC 152.2/8H5 Catalog #359-020, Ancell Corp. Bayport, Mn.

Example 8 10D1 Binds to Human Activated T Cells

The ability of 10D1 antibody to bind to CTLA-4 expressed by normal humanT cells was investigated by flow cytometric analysis of resting andactivated T cells (FIG. 14). Freshly isolated human peripheral bloodmononuclear cells at 2×10⁶/ml were incubated in the presence or absenceof 2 ug/ml of the T-cell mitogen, phytohemagglutinin (PHA). After fourdays incubation, the cells were washed and stained with the followingantibodies: 1) no antibody; 2) HuIgG1-FITC, a human IgG1 anti EGFreceptor antibody; 3) 10D1-FITC, human IgG1 antiCTLA-4 antibody; and 4)147-FITC—mouse anti-human CTLA-4 antibody. After incubation for 1 hr.,cells were washed and stained with rabbit anti-FITC IgG followed by goatanti-rabbit-PE. Analysis was performed on lymphocytes gated by forwardversus side scatter. As shown in FIG. 14, resting lymphocytes do notbind 10D1 antibody, while PHA-activated T cells express low levels ofCTLA-4 at the cell surface

Example 9 10D1 Does not Mediate Complement-Dependent orAntibody-Dependent Lysis of Activated T-cells

The ability of MAb 10D1 to mediate complement-dependent cellularcytotoxicity (CDCC) or antibody-dependent cellular cytotoxicity (ADCC)of CTLA-4 expressing cells was investigated.

For CDCC experiments, rabbit serum was used as a source of compliment,in order to provide optimal conditions for CDCC. Rabbit complement hasbeen shown to be more effective in mediating CDCC with human IgG₁ thanhuman complement (Jurianz, Maslak et al., 1999). PHA-stimulated T-cellswere labeled with ⁵¹Cr and incubated with various concentrations ofanti-CTLA4 MAb 10D1 or anti-CD3 MAb with or without rabbit serum as asource of complement. After a 1 hour incubation, the ⁵¹Cr released bydying cells was determined using a gamma counter. Target cells incubatedwith 2% SDS served as 100% lysis controls. The anti-CTLA-4 MAb 10D1 didnot mediate CDCC of the activated T-cells (FIG. 15). Under the sameconditions, the murine IgG2, anti-CD3 MAb led to significant CDCC. Bothmurine IgG2_(a) and human IgG₁ efficiently fix rabbit complement;therefore these differences most likely reflect the greatly reducedexpression of CTLA-4 as compared to CD3 on activated T-cells.

Similarly, no ADCC activity was observed for MAb 10D1 using autologousmononuclear cells as effector cells (FIG. 16). PHA-stimulated T-cellswere labeled with ⁵¹Cr and incubated with various concentrations ofanti-CTLA4 MAb 10D1 or anti-CD3 MAb and fresh autologous mononuclearcells. The effector to target cell ratio was 100:1. After a 4 hourincubation, the ⁵¹Cr released by dying cells was determined using agamma counter. Target cells incubated with 2% SDS served as 100% lysiscontrols. Although the anti-CD3 MAb is a murine IgG2_(a), which canmediate efficient ADCC with human effector cells, only low levels ofADCC were observed. These data are consistent with the requirement ofhigh levels of antigen expression on the surface of target cells forefficient ADCC. Since MAb 10D1 is a human IgG₁, an isotype generallycapable of mediating CDCC and ADCC, the lack of these activities islikely due to the very low expression of CTLA-4 on activated T-cells.Furthermore, the observation of increased numbers of activated T-cellsin the primate toxicology studies (see below) is consistent with thelack of ADCC and CDCC activity of activated T-cells by MAb 10D1 in vivo.

Example 10 10D1 Preclinical Toxicity Studies in Cynomolaus Monkeys

Two independent toxicology studies of 10D1 antibody and macaques wereperformed. A total of eight monkeys were analyzed. Four monkeys (twomales and two females) tolerated three bolus i.v. doses of 3 mg/Kg humananti-CTLA4, and four monkeys (two males and two females) tolerated threebolus i.v. doses of 10 mg/Kg human anti-CTLA4 without significantclinical, immunotoxicology, or histopathological findings.

A. 10D1 Primate Toxicology Study (3.0 mg/Kg)

To investigate the effects of 10D1 in vivo, a primate toxicology studywas performed with two macaques. In a multiple dose toxicity study ofMAb 10D1, this antibody was administered via intravenous injection ofmacaques. The objective of this study was to determine the tolerabilityof MAb 10D1 in two monkeys given at a dose and schedule compatible withefficacious treatment in a murine tumor regression model and proposeddose in human clinical studies. Two female cynomolgus monkeys (Macacafascicilaris) were treated with three intravenous bolus doses of 3.0mg/Kg 10D1 on days 1, 4, and 7 to evaluate safety and T-cell activationin these animals. The animals were observed for any adverse reactions,weight loss/gain, and morbidity and mortality up to 14 days postadministration of the first dose. Seven days after the last dose theanimals were sacrificed and necropsied to examine their organsindividually. Blood samples were collected before each dose and beforenecropsy for examination of T-cell populations and expression ofactivation markers by flow cytometry. Plasma was also collected fromblood samples to determine 10D1 antibody levels and anti-10D1 antibodyresponses by ELISA.

The animals tolerated three doses of antibody 10D1 without any clinicalsymptoms during the treatment course. The weight of these animals didnot change significantly. No gross findings were documented on 47organs/tissues examined at necropsy for either animal.

Histopathology studies were performed at Redfield laboratories,Redfield, Ark. The results from these studies indicated that multipledoses of MAb 10D1 did not produce acute toxicity in any of the organsand tissues examined.

Pharmacokinetic analysis revealed the presence of significant levels (upto 97.3 μg/ml) of 10D1 MAb in the plasma of both monkeys (see Table 7).Plasma levels of 10D1 were determined by a competition assay withFITC-10D1 using flow cytometry and 58αβCTLA-4 T-cells.

TABLE 7 10D1 plasma levels Time point Monkey #1 Monkey #2 Pre-1^(st)dose  0.0 (μg/ml plasma)  0.0 (μg/ml plasma) Day 4, pre-2^(nd) dose 17.4(μg/ml plasma) 43.6 (μg/ml plasma) Day 7, pre-3^(rd) dose 83.6 (μg/mlplasma) 97.3 (μg/ml plasma) Day 14 90.2 (μg/ml plasma) 70.9 (μg/mlplasma)

Evaluation of the anti-10D1-antibody response was performed by ELISA. Nosignificant anti-10D1 response was observed in either animal during thecourse of study (FIG. 17). Microtiter plates were coated with 10D1 MAb(for IgM assay) or 10D1 F(ab′)₂ (for IgG assay). Dilutions of plasmasamples from various time points were incubated with the plates, andanti-10D1 antibodies were detected with either anti-IgM or IgGFe-specific alkaline phosphatase reagents. IgM anti-10D1 antibodiesappear to have developed by day 14, however, the titers are very low.IgM anti-10D1 antibodies appear to have developed by day 14, however,the titers are very low. These data demonstrate that the monkeys did notdevelop anti-10D1 antibody responses after 3 doses of the antibody.

These data demonstrate that the animals did not develop a significantantibody response against MAb 10D1 during the course of this study.

Immunotoxicology was investigated by flow cytometric analysis oflymphocyte populations during the course of the study. The lymphocytesubsets examined included CD3 as a marker for total T-cells and CD20 asa marker for total B-cells. T-cells, were further subdivided forexpression of CD4 (helper T-cell marker) and CD8 (cytotoxic T-cellmarker), as well as for activation markers CD25, CD29, CD69 and HLA-DR.No remarkable changes in T-cell populations or expression of activationmarkers was noted. The results are summarized in Table 8 below.

TABLE 8 Flow cytometric analysis of lymphocyte populations Time pointMonkey #1 Monkey #2 Pre-1^(st) dose % CD3 = 61, % CD20 = 16 % CD3 = 54,% CD20 = 22 % CD4 = 43, % CD8 = 50 % CD4 = 59, % CD8 = 36 % CD25 ≦ 1, %CD29 = 41 % CD25 ≦ 1, % CD29 = 29 % CD69 = <1, % HLA-DR = 4 % CD69 ≦ 1,% HLA-DR = 1 Day 4, pre-2^(nd) dose % CD3 = 58, % CD20 = 13 % CD3 = 56,% CD20 = 16 % CD4 = 38, % CD8 = 52 % CD4 = 62, % CD8 = 37 % CD25 ≦ 1, %CD29 = 52 % CD25 ≦ 1, % CD29 = 36 % CD69 ≦ 1, % HLA-DR = 2 % CD69 ≦ 1, %HLA-DR ≦ 1 Day 7, pre-3^(rd) dose % CD3 = 59, % CD20 = 15 % CD3 = 51, %CD20 = 17 % CD4 = 47, % CD8 = 59 % CD4 = 51, % CD8 = 39 % CD25 = 2, %CD29 = 44 % CD25 = 1, % CD29 = 39 % CD69 = 1, % HLA-DR = 4 % CD69 = 1, %HLA-DR = 2 Day 14 % CD3 = 64, % CD20 = 14 % CD3 = 59, % CD20 = 20 % CD4= 49, % CD8 = 44 % CD4 = 60, % CD8 = 35 % CD25 = 1, % CD29 = 44 % CD25 ≦1, % CD29 = 34 % CD69 ≦ 1, % HLA-DR = 15 % CD69 ≦ 1, % HLA-DR = 1

Heparinized blood samples were analyzed fresh by flow cytometry usingFITC- or PE-labeled anti-lymphocyte reagents. % CD3 and % CD20 are basedon a lymphocyte gate. The additional T-cell markers and activationmarkers are all based on CD3-positive cells. These data indicate thatmultiple doses of MAb 10D1 does not have a significant effect on B andT-cell populations or T-cell activation markers.

B. 10D1 Primate Toxicology Study (3.0 and 10.0 mg/Kg)

Six cynomolgus monkeys (four males and two females), experimentallynon-naïve and weighing 2.4 to 3.8 kg at the outset of the study, wereassigned to treatment groups as shown in Table 9 below.

TABLE 9 Number of Dose Level Dose Vol. Dose Solution Group No.Males/Females (mg/kg) (ml/kg) Conc. mg/ml) 1 2/0 3 0.6 5.0 2 2/2 10 2.05.0

Each animal received a dose of human anti-CTLA4 (5 mg/ml concentration)by intravenous injection (i.e., “slow-push” bolus injection) every threedays for one week (i.e., on Days 1, 4 and 7). Detailed clinicalobservations were conducted at least twice daily (“cagesideobservations”), and a thorough physical examination was performed oneach animal prior to the study and on Day 12. Body weights were measuredweekly (prestudy and Days 7 and 14), and ophthalmoscopic examination wasconducted on all animals prior to the study and on Day 12. Blood samplesfor evaluation of serum chemistry, hematology and coagulation parameterswere collected from all animals prestudy and on Day 14. Additionalsamples for selected hematology parameters (total and differential whiteblood cells only) were collected prior to dosing on each dosing day(Days 1, 4, and 7). Urine samples for standard urinalysis were obtainedby drainage from specially designed cage-pans prior to dosing and on Day13. Blood samples were also collected prior to each dose (Days 1, 4 and7) and prior to termination (Day 14) for various analyses conducted byMedarex. These included analysis of test article concentration(pharmacokinetics), determination of the presence of antibodies to thetest article, and flow cytometry analysis. All animals were euthanizedon Day 14, at which time, a complete gross necropsy was conducted, majororgans were weighed, and a standard complete set of tissues wascollected from each animal and processed for examination by lightmicroscopy.

Intravenous administration of human anti-CTLA4 at dose levels of 3 mg/kgand 10 mg/kg given every three days for a total of three doses was verywell tolerated by cynomolgus monkeys. There were no clinical signs oftoxicity from the cageside observations and physical examinations, andno effects on body weight, ocular examination findings, clinicalpathology parameters, gross necropsy findings, organ weights or tissuehistomorphology.

The results of the analysis of test article concentration in serumsamples (i.e., trough levels measured in samples obtained prior todosing on Days 4 and 7, and prior to necropsy on Day 14) indicateddose-dependent exposure to the test article. On Day 7, predose meanconcentrations were approximately 84 and 240 μg/ml for the 3- and10-mg/kg dose groups, respectively.

A potential for accumulation of the test article in serum with theevery-three-day dosing schedule in monkeys was evident from thedifference between the Day 4 and Day 7 trough levels (i.e., meansconcentrations on Day 7 were approximately twice as high as on Day 4),as well as from the high residual levels on Day 14 (one week after thelast dose), which were similar to the Day 7 trough levels. Evidence ofantibody formation against the test article was detected in two of thesix study animals (one from Group 1 and another from Group 2). In theformer case, it appeared that the antibody response might have affectedthe clearance of the test article from circulation. Flow cytometricanalysis of lymphocyte subsets revealed a modest increase in totalCD3-positive cells between Days 1 and Day 14, which correlated with anincrease in CD3/CD4-positive cells, and a respective decrease inCD3/CD8-positive cells (Group 2 only). The percentage of CD3 cellsexpressing CD29 and HLA-DR moderately increased over the course of thestudy, which was consistent with previous findings that anti-CTLA4antibodies can enhance antigen-specific T-cells.

In conclusion, apart from the minor changes in circulating lymphocytesubpopulations, the highest dose level tested in this study (i.e., threedoses of 10 mg/kg given at three-day intervals) was an absoluteno-effect dose level in cynomolgus monkeys.

Example 11 A Phase I Human Clinical Trial of MAb 10D1 in Prostate Cancer(MDXCTLA4-01) and Melanoma (MDXCTLA4-02)

MDXCTLA4-01 is an open-label study of anti-cytotoxicT-lymphocyte-associated antigen-4 (anti-CTLA-4) monoclonal antibody 10D1(MAb 10D1) in patients with progressive, metastatic, hormone-refractoryprostate cancer. Treatment is a single dose of MAb 10D1 that isadministered intravenously, as an infusion, at a dosage of 3.0 mg/Kg.

The objectives of this trial are to determine if i. administration ofMAb 10D1 causes nonspecific T-cell activation, ii. to establish asafety/tolerability profile for MAb 10D1 in these patients and, iii. todetermine the pharmacokinetic profile of MAb 10D1 and assess thedevelopment of a host immune response to MAb 10D1. In addition the studywill attempt to identify preliminary evidence of efficacy. The study isa multicenter, open-label study of a single dose of MAb 10D1 in 14subjects. The study consists of four phases: Screening, Infusion,Post-infusion, and Follow-up (see Table 10 below).

TABLE 10 Follow- Phase Screen Infusion Post-infusion up Time days −30 to130 145 160 190 250 370 24 48 72 day day day day monthly −14 to 0 minmin min min min min hrs his hrs 7 14 21 28

Patients with histologic diagnosis of primary adenocarcinoma of theprostate, and progressive metastatic carcinoma of the prostate afterandrogen deprivation and at least one systemic non-hormonalmanipulation, are being screened for participation in this study.Subjects must have progressive measurable disease, progressive PSA,PSA>5 ng/ml, testosterone<50 ng/dl, primary gonadal androgensuppression, life expectancy>12 weeks, and Karnofsky PerformanceStatus≧60%.

Subjects undergo physical examination, ECG, chest radiography,diagnostic imaging, and blood sampling for hematological, biochemical,and immune function assessments, and have vital signs monitored. Monthlytelephone interviews are used to collect and record information on asubset of adverse events, including autoimmune adverse events afterdisease progression, until six months after treatment. PSA (decline,duration of decline, progression, time to progression) and diseaseresponse (complete, partial, stable, progressive) are monitored. Plasmaconcentrations of MAb 10D1 are being assessed immediately prior to,during, and up to two months after, infusion.

Data from four prostate cancer subjects that have been treated are shownin Table 11. No adverse events have been recorded. For all of thesubjects treated, MAb 10D1 appears to be well tolerated.

Because of the importance of monitoring the immune status of patients inthe trial and the specific goal of monitoring generalized effects on Tcell activation by anti-CTLA-4 antibody, the entry criteria in thisstudy included minimum levels of CD4 and CD8 T cells of 500/ml and≧500/ml respectively. However, it was observed during the initialaccrual in the study that prostate cancer patients have significantlyreduced T cell numbers although CD4 and CD8 T cells are clearly present.Many patients were initially rejected based on the above entry criteria(see Table 11). The apparent reduced T cell counts observed is apreviously undocumented observation in prostate cancer patients that mayhave relevance in treatments involving cancer vaccination in thesepatients. Subsequent to these observations, the entry criteria wereamended to include patients having CD4 and CD8 count of ≧300/ml and≧200/ml respectively.

In order to evaluate whether administration of MAb 10D1 can induceundesirable non-specific T cell activation, peripheral blood lymphocytesfrom the prostate cancer subjects were analyzed by flow cytometry foreach of the following markers: CD4, CD8, CD25, CD44, CD69 and HLA-DR.Blood samples were taken at time points indicated in Table 10. Nosignificant change in the frequency of any of these markers was observedduring the course of the treatment for each of the prostate cancersubjects treated thus far. An example of this analysis is shown in Table12 which shows the frequency of CD4, CD25, CD69-positive cells and CD8,CD25, CD69-positive cells at times prior to, during, and subsequent toMAb 10D1 administration in two of the subjects. These data demonstratethat MAb 10D1 does not result in non-specific T cell activation.

TABLE 11 Study No. MDXCTLA4-01 Selected Lab Values Summary ScreenSubject Amend- PSA Platelets WBC Neuts Lymphs no. no. Initials ment #Day Date ng/ml ×10³/ul ×10³/ul % ×10³/ul % ×10³/ul 02001 001 JGR Scr144.80 263 8.12 73.00 5.90 18.00 1.47 02001 001 JGR 0 185.20 267 5.7466.00 3.79 22.00 1.32 02001 001 JGR 1 259 6.31 69.00 4.38 20.00 1.2902001 001 JGR 2 240 6.59 70.00 4.66 19.00 1.31 02001 001 JGR 3 270 6.5371.00 4.63 21.00 1.36 02001 001 JGR 7 257.40 299 6.70 68.00 4.56 23.001.53 02001 001 JGR 14  332.30 308 6.87 71.90 7.94 21.20 1.39 02001 001JGR 21  286 9.72 74.00 7.20 19.70 1.91 02001 001 JGR 28  351.00 304 5.3863.00 3.40 26.00 1.44 01002 JWF Scr  28.30 271 11.60  75.40 8.75 13.601.58 01003 MZB Scr  12.70 178 5.49 69.00 3.79 19.60 1.08 01004 TEQ Scr1459.00  264 6.26 75.10 4.70 14.40 0.90 01005 WMN Scr 192.40 212 6.8573.70 5.05 17.40 1.20 01006 MRS Scr 4503.00  140 7.55 76.70 5.79 15.901.20 01007 TAB Scr 1394.00  205 5.78 73.00 4.24 13.00 0.76 01008 CHB Scr 70.70 229 4.67 54.00 2.56 32.00 1.52 01009 003 RAB Scr 238.60 144 3.7078.00 2.88 14.00 0.55 01009 003 RAB 0 336.90 123 3.92 68.00 2.67 21.000.83 01009 003 RAB 1 122 3.35 71.00 2.38 22.00 0.74 01009 003 RAB 2 1094.06 74.00 2.99 19.00 0.77 01009 003 RAB 3 114 3.79 70.00 2.67 21.000.81 01009 003 RAB 7 249.30  69 3.38 75.00 2.54 17.00 0.60 01009 003 RAB14  269.80 101 3.68 69.00 2.54 21.20 0.78 01009 003 RAB 21  122 4.8278.00 3.76 13.20 0.64 01012 004 CEH Scr 112.90 172 5.85 64.00 3.74 28.001.69 01012 004 CEH 1 01012 004 CEH 2 150 4.82 67.70 3.26 28.40 1.2801012 004 CEH 3 147 4.36 63.70 2.78 29.30 1.28 01012 004 CEH 7 190.00159 4.95 58.60 2.90 32.70 1.61 01012 004 CEH 14 207.60 199 5.64 63.103.55 29.30 1.65 01013 KJF Scr  49.10 228 8.53 65.00 5.62 26.00 2.2302014 002 L-S Scr  12.70 222 5.65 53.00 3.01 34.00 1.92 02014 002 L-S 0 27.50 217 5.88 57.00 3.36 32.00 1.88 02014 002 L-S 1 226 5.74 55.003.19 35.00 2.04 02014 002 L-S 2 223 5.59 55.00 3.09 32.00 1.84 02014 002L-S 3 219 4.89 54.00 2.66 34.00 1.68 01016 Ineligible G-F Scr 4856.00 106 7.31 86.00 6.29  5.00 0.33 normal range low 150 3.80 40.50 1.9615.40 0.80 high  7.00 10.70  75.00 7.23 48.50 3.00 Screen Monos Eos CD4/CD8/ ESR Hgb Hcrit no. % ×10³/ul % ×10³/ul ul ul mm/hr g/dl % 02001 5.600.46 1.80 0.15 670 367 71 10.4 30 02001 6.60 0.38 3.10 0.18 704 376 10.632 02001 8.70 0.55 0.90 0.00 A A  9.5 30 02001 6.70 0.44 1.80 0.12 556303  9.5 28 02001 5.50 0.36 2.20 0.14 608 254  9.3 28 02001 6.00 0.462.50 0.17 A A  9.5 28 02001 5.21 0.36 1.90 0.13 A A  8.8 25 02001 4.800.46 1.00 0.10 A A  9.1 28 02001 5.80 0.31 2.90 0.16  8.7 25 01002 5.700.66 4.60 0.53 399 189 41 13.9 37 01003 6.30 0.35 2.70 3.24 325 168 1912.7 36 01004 7.70 0.48 2.40 0.15 365 129 61 12.8 36 01005 6.20 0.432.20 0.15 483 217 01006 6.20 0.47 0.80 0.06 319 363 83 01007 6.50 0.376.00 0.35 376 127 14.1 43 01008 8.30 0.39 3.40 0.16 461 499 15.6 4501009 5.40 0.20 1.20 0.04 211 162 43  9.8 30 01009 8.70 0.34 1.50 0.06374 188 10.9 31 01009 4.00 0.14 1.80 0.06 307 192 11.3 32 01009 4.800.20 1.20 0.05 328 220 11.3 33 01009 6.20 0.23 1.30 0.05 313 266 10.9 3101009 5.60 0.19 0.70 0.02 244 161 10.4 30 01009 8.50 0.31 1.00 0.04 308173  8.8 25 01009 7.70 0.37 0.60 0.03 218 195  7.4 20 01012 5.60 0.331.00 0.06 746 451 10 13.2 40 01012 642 475 01012 4.60 0.22 1.10 0.05 552380 12.2 36 01012 5.10 0.22 1.30 0.06 544 441 13.1 37 01012 5.90 0.292.50 0.12 842 506 12.6 35 01012 5.70 0.32 1.60 0.09 13.5 38 01013 5.300.46 2.30 0.20 1213  398 13.4 37 02014 7.40 0.42 3.90 0.22 721 439 13.640 02014 8.60 0.50 1.50 0.09 676 389 13.5 38 02014 7.00 0.40 1.40 0.08632 405 13.6 38 02014 9.80 0.55 1.40 0.09 590 339 13.5 39 02014 7.500.37 2.70 0.13 529 358 13.2 37 01016 6.80 0.49 1.90 0.14   57 6  7 10.331 2.60 0.12 404 220 10.00  0.92 6.80 0.57 1612  1128  30

TABLE 12 Flow cytometric analysis of T cell activation markers inprostate cancer subjects treated with 3.0 mg/Kg MAb 10D1. Patient NumberTime Point CD (4 + 25 + 69) % CD (8 + 25 + 69) % 3 Screen 1.7 0.8 3 −30MIN (Pre- 2.6 0.8 Infusion) 3  40 MIN 2.5 0.7 3 130 MIN 1.9 0.9 3 145MIN 1.7 0.5 3 160 MIN 1.7 1 3 190 MIN 1.5 1.5 3 250 MIN 2.1 1.2 3 370MIN 1.3 0.9 3 24 HR 1.6 1.6 3 48 HR 2.7 3 3 72 HR 0.9 0.5 3 Day 7  0.90.1 3 Day 14 0.4 0.5 3 Day 21 2.3 1.9 4 Screen 1.4 0.8 4 −30 MIN (Pre-0.5 0.3 Infusion) 4  40 MIN 0.3 0.1 4 130 MIN 0.3 0.1 4 145 MIN 0.4 0.24 160 MIN 0.2 0.2 4 190 MIN 0.8 0.3 4 250 MIN 0.1 0 4 370 MIN 0.3 0.1 424 HR 0.2 0.3 4 48 HR 0.4 0.6 4 72 HR 0.8 0.3 4 Day 7  1 0.7 4 Day 141.1 0.8

A second clinical trial (MDXCTLA4-02) using MAb 10D1 in subjects withStage 1V malignant melanoma has also been initiated. A single dose ofMAb 10D1 will be administered intravenously, as an infusion, at a dosageof 3.0 mg/Kg. This study also consists of four phases (Screening,Infusion, Post-Infusion and Follow-up) as described in Table 9, above.

The goals of this study are as those regarding the above-described studyin prostate cancers as well as to specifically establish asafety/tolerability profile for MAb 10D1 in patients with Stage 1Vmalignant melanoma. One patient has been treated in this study (seeTable 13). As in the prostate cancer study, MAb 10D1 appears to be welltolerated. Flow cytometric analysis of T cell activation markers in thissubject, analogous to that performed for the prostate tumor trial, alsoshowed no evidence of non-specific T cell activation,

TABLE 13 Study No. MDXCTLA4-02 Selected Lab Values Summary Amend-Platelets WBC Neuts Lymphs Monos Screen no. Subject no. Initials ment #Day Date ×10³/ul ×10³/ul % ×10³/ul % ×10³/ul % ×10³/ul 02001 001 SAH 0Scr 216 6.28 56.60 3.52 35.60 2.23 5.90 0.37 02001 001 SAH 0 0 230 5.5859.70 3.33 32.30 1.80 5.70 0.32 02001 001 SAH 0 1 202 5.12 61.80 3.1630.20 1.55 5.00 0.26 normal range low 150 3.80 40.50 1.96 15.40 0.802.60 0.12 high 10.70  75.00 7.23 48.50 3.00 10.10  0.92 Amend- Eos CD4/CD8/ ESR Hgb Hcrit Screen no. Subject no. Initials ment # Day Date %×10³/ul ul ul mm/hr g/dl % 02001 001 SAH 0 Scr 1.80 0.11 1189 631 14.439 02001 001 SAH 0 0 1.80 0.10 1039 502 14.9 43 02001 001 SAH 0 1 2.300.12  957 407 13.4 37 normal range low  404 220 high 6.80 0.57 16121129  30

Ongoing results from the MDXCTLA4-0 I and MDXCTLA4-02 clinical trialshave demonstrated that the infusions are tolerable with only minorreactions. Prolonged plasma half-life of the antibody was seen, with theantibody remaining in the plasma for approximately 3 to 4 months. Clearevidence of immune effects was observed without overt non-specific Tcell activation. Symptomatic relief and reductions in prostate specificantigen (PSA) levels have been observed in prostate cancer patientstreated with the anti-CTLA-4 antibody. Representative results forreductions in PSA levels are shown in FIG. 18, which shows PSA levels(in ng/ml) in two patients (one represented by the closed circles, theother by the open circles) at various time points after infusion of 3mg/kg anti-CTLA-4 antibody at day 0. The results demonstrate that PSAlevels decreased after infusion of the antibody and remained suppressedfor approximately 3-4 months after treatment, correlating with thepresence of the anti-CTLA-4 antibody in the plasma. Other examples ofimmune effects observed included immune-mediated rash and pruritis,transient seroconversion to positive autoantibodies, melanin pigmentchanges in melanoma patients and inflammatory reactions at tumor sites.Except for the rash and pruritis, all potentially adverse immune effectswere subclinical. In summary, the ongoing results from human clinicaltrials with anti-CTLA-4 antibody treatment demonstrate that the antibodyis well-tolerated and stimulates immune effects in recipients.

Example 12 Anti-CTLA-4 Treatment Enhances Antibody Responses to aHepatitis B Surface Antigen (HBsAg) Vaccine

The ability of a human anti-CTLA-4 antibody of the invention to enhanceantibody responses to a hepatitis B surface antigen (HBsAg) vaccine wasexamined in cynomolgus monkeys. Test groups of four monkeys each (twomales, two females) were treated with either 1) the HBsAg vaccine incombination with a control IgG1 antibody (a humanized anti-RSV antibody,Synagis™, commercially available from MedImmune) or 2) the HbsAg vaccinein combination with the anti-CTLA-4 antibody 10D1. The anti-CTLA-4antibody or control IgG1 were administered intravenously at a dosage of10 mg/kg in a volume of 2.0 ml/kg. The HBsAg vaccine (Engerix-B™,commercially available from GlaxoSmithKline) was administeredintramuscularly at a dosage of 10 mg in a volume of 0.5 ml. Theanti-CTLA-4 or control IgG1 antibody was administered on days 1 and 29,whereas the HBsAg vaccine was administered on days 2 and 30. Plasmalevels of anti-HBsAg antibody were measured on days 1, 51 and 64 using aradioimmunoassay kit (commercially available from Abbott). Resultspresented represent the mean of the four animals in each group, +/−SE.The results are shown in the bar graphs of FIG. 19, wherein Group 1 wastreated with vaccine and control IgG1 and Group 2 was treated withvaccine and anti-CTLA-4. The left bar for each group represents day 1,the middle bar represents day 51 and the right bar represents day 64.Use of the anti-CTLA-4 antibody in combination with the vaccine led to asignificantly greater anti-HBsAg antibody response than use of thevaccine with a control IgG1. These results demonstrate that a humananti-CTLA-4 antibody of the invention is capable of enhancing antibodyresponses to a viral antigen vaccine in vivo in primates.

Example 13 Anti-CTLA-4 Treatment Enhances Antibody and T Cell Responsesto a Melanoma Cell Vaccine

The ability of a human anti-CTLA-4 antibody of the invention to enhanceantibody and T cell responses to a melanoma cell vaccine was examined incynomolgus monkeys. Test groups of six monkeys each (three males, threefemales) were treated with either 1) a melanoma cell vaccine alone(SK-mel-3, a human melanoma tumor cell line transfected to expressGM-CSF) or 2) both SK-mel-3 and the anti-CTLA-4 antibody 10D1. Theantibody was administered intravenously at a dosage of 10 mg/kg in avolume of 1.3 ml/kg. The SK-mel-3 cells were administered subcutaneouslyin a fixed amount (5×10⁶ cells/animal at 0.5 ml/animal). The appropriateantibody and/or vaccine were administered on days 0, 28, 56 and 84.Antibody responses to the melanoma cell vaccine were assessed on days13, 41, 69 and 97. The results are shown in the graph of FIG. 20, inwhich a 1/1000 dilution of plasma was examined. Results presentedrepresent the mean of the six animals in each group, +/−SE. Results fromthe animals treated with the vaccine alone are depicted with the opencircles, whereas results from animals treated with both the anti-CTLA-4antibody and the vaccine are depicted with closed circles. Use of theanti-CTLA-4 antibody in combination with the vaccine led to asignificantly greater antibody response against the melanoma cells thanuse of the vaccine alone. These results demonstrate that a humananti-CTLA-4 antibody of the invention is capable of enhancing antibodyresponses to a tumor cell vaccine in vivo in primates.

The effect of anti-CTLA-4 treatment on antigen-specific T cellproliferation was also examined. Prior to vaccination of the animals,blood was drawn and monocytes from the animals were differentiated invitro into dendritic cells (DC) to provide a population of autologousdendritic cells for use in T cell proliferation studies. A portion oftheses autologous DCs were incubated with the SK-mel-3 cells to providea population of autologous DCs that had been pulsed with melanomaantigens. At various time points after vaccination, polymorphonuclearcells (PMNC) were obtained from the animals and incubated in vitroeither 1) alone (as a negative control), 2) with Staphylococcusenterotoxin B (SEB, a non-specific activator of certain T cellpopulations, as a positive control), 3) with autologous dendritic cellsor 4) with autologous dendritic cells that had been pulsed with melanomaantigens. T cell proliferation was assessed using a quantitative flowcytometry assay that allowed for a quantitative measure of the totalnumber of T cells per well (through the use of an anti-CD3 antibody), aswell as the number of CD8⁻ vs. CD8⁺ cells (through the use of ananti-CD8 antibody). The results from an animal treated with SK-mel-3 incombination with anti-CTLA-4, assessed at day 41 after vaccination, aresummarized in FIG. 21, wherein T cell proliferation is expressed as astimulation index relative to the number of control unstimulated cells(set at a stimulation index of one). As illustrated in FIG. 21,stimulation with the non-specific activator SEB increased thestimulation index at least 5 fold in both CD8⁺ and CD8⁻ cells, whereasincubation with autologous dendritic cells alone increased thestimulation index only very slightly. Incubation with autologousdendritic cells pulsed with melanoma cell antigens also increased thestimulation index at least 5 fold in both CD8⁺ and CD8⁻ cells (thelatter essentially corresponding to the CD4⁺ T cell population), therebyindicating that vaccination with the melanoma cell vaccine incombination with anti-CTLA-4 results in antigen-specific T cellproliferation of both CD8⁺ and CD4⁺ T cells.

Further evidence of antigen-specific T cell proliferation was obtainedfrom delayed type hypersensitivity (DTH) experiments. Animals treatedwith either the melanoma vaccine alone or with the melanoma vaccine incombination with the anti-CTLA-4 antibody were tested for a DTH reactionto either SK-mel-3 or to a saline control using standard DTH assaymethods. The results demonstrated that 3 of 6 of the animals treatedwith the combination of the vaccine and the anti-CTLA-4 antibodyexhibited a specific DTH response to the SK-mel-3 cells, whereas onlyone of the 6 animals treated with the vaccine alone exhibited a specificDTH response to the SK-mel-3 cells. These results further demonstratethe ability of anti-CTLA-4 antibody treatment to enhanceantigen-specific T cell responses in vivo in primates.

Although the foregoing invention has been described in detail forpurposes of clarity of understanding, it will be obvious that certainmodifications may be practiced within the scope of the appended claims.All publications and patent documents cited herein are herebyincorporated by reference in their entirety for all purposes to the sameextent as if each were so individually denoted.

APPENDIX SEQUENCE LISTING SEQ ID NO: 1 pGPlkAATTAGCGGC CGCTGTCGAC AAGCTTCGAA TTCAGTATCG ATGTGGGGTA   50CCTACTGTCC CGGGATTGCG GATCCGCGAT GATATCGTTG ATCCTCGAGT  100GCGGCCGCAG TATGCAAAAA AAAGCCCGCT CATTAGGCGG GCTCTTGGCA  150GAACATATCC ATCGCGTCCG CCATCTCCAG CAGCCGCACG CGGCGCATCT  200CGGGCAGCGT TGGGTCCTGG CCACGGGTGC GCATGATCGT GCTCCTGTCG  250TTGAGGACCC GGCTAGGCTG GCGGGGTTGC CTTACTGGTT AGCAGAATGA  300ATCACCGATA CGCGAGCGAA CGTGAAGCGA CTGCTGCTGC AAAACGTCTG  350CGACCTGAGC AACAACATGA ATGGTCTTCG GTTTCCGTGT TTCGTAAAGT  400CTGGAAACGC GGAAGTCAGC GCCCTGCACC ATTATGTTCC GGATCTGCAT  450CGCAGGATGC TGCTGGCTAC CCTGTGGAAC ACCTACATCT GTATTAACGA  500AGCGCTGGCA TTGACCCTGA GTGATTTTTC TCTGGTCCCG CCGCATCCAT  550ACCGCCAGTT GTTTACCCTC ACAACGTTCC AGTAACCGGG CATGTTCATC  600ATCAGTAACC CGTATCGTGA GCATCCTCTC TCGTTTCATC GGTATCATTA  650CCCCCATGAA CAGAAATTCC CCCTTACACG GAGGCATCAA GTGACCAAAC  700AGGAAAAAAC CGCCCTTAAC ATGGCCCGCT TTATCAGAAG CCAGACATTA  750ACGCTTCTGG AGAAACTCAA CGAGCTGGAC GCGGATGAAC AGGCAGACAT  800CTGTGAATCG CTTCACGACC ACGCTGATGA GCTTTACCGC AGCTGCCTCG  850CGCGTTTCGG TGATGACGGT GAAAACCTCT GACACATGCA GCTCCCGGAG  900ACGGTCACAG CTTGTCTGTA AGCGGATGCC GGGAGCAGAC AAGCCCGTCA  950GGGCGCGTCA GCGGGTGTTG GCGGGTGTCG GGGCGCAGCC ATGACCCAGT 1000CACGTAGCGA TAGCGGAGTG TATACTGGCT TAACTATGCG GCATCAGAGC 1050AGATTGTACT GAGAGTGCAC CATATGCGGT GTGAAATACC GCACAGATGC 1100GTAAGGAGAA AATACCGCAT CAGGCGCTCT TCCGCTTCCT CGCTCACTGA 1150CTCGCTGCGC TCGGTCGTTC GGCTGCGGCG AGCGGTATCA GCTCACTCAA 1200AGGCGGTAAT ACGGTTATCC ACAGAATCAG GGGATAACGC AGGAAAGAAC 1250ATGTGAGCAA AAGGCCAGCA AAAGGCCAGG AACCGTAAAA AGGCCGCGTT 1300GCTGGCGTTT TTCCATAGGC TCCGCCCCCC TGACGAGCAT CACAAAAATC 1350GACGCTCAAG TCAGAGGTGG CGAAACCCGA CAGGACTATA AAGATACCAG 1400GCGTTTCCCC CTGGAAGCTC CCTCGTGCGC TCTCCTGTTC CGACCCTGCC 1450GCTTACCGGA TACCTGTCCG CCTTTCTCCC TTCGGGAAGC GTGGCGCTTT 1500CTCATAGCTC ACGCTGTAGG TATCTCAGTT CGGTGTAGGT CGTTCGCTCC 1550AAGCTGGGCT GTGTGCACGA ACCCCCCGTT CAGCCCGACC GCTGCGCCTT 1600ATCCGGTAAC TATCGTCTTG AGTCCAACCC GGTAAGACAC GACTTATCGC 1650CACTGGCAGC AGCCAGGCGC GCCTTGGCCT AAGAGGCCAC TGGTAACAGG 1700ATTAGCAGAG CGAGGTATGT AGGCGGTGCT ACAGAGTTCT TGAAGTGGTG 1750GCCTAACTAC GGCTACACTA GAAGGACAGT ATTTGGTATC TGCGCTCTGC 1800TGAAGCCAGT TACCTTCGGA AAAAGAGTTG GTAGCTCTTG ATCCGGCAAA 1850CAAACCACCG CTGGTAGCGG TGGTTTTTTT G1TTGCAAGC AGCAGATTAC 1900GCGCAGAAAA AAAGGATCTC AAGAAGATCC TTTGATCTTT TCTACGGGGT 1950CTGACGCTCA GTGGAACGAA AACTCACGTT AAGGGATTTT GGTCATGAGA 2000TTATCAAAAA GGATCTTCAC CTAGATCCTT TTAAATTAAA AATGAAGTTT 2050TAAATCAATC TAAAGTATAT ATGAGTAAAC TTGGTCTGAC AGTTACCAAT 2100GCTTAATCAG TGAGGCACCT ATCTCAGCGA TCTGTCTATT TCGTTCATCC 2150ATAGTTGCCT GACTCCCCGT CGTGTAGATA ACTACGATAC GGGAGGGCTT 2200ACCATCTGGC CCCAGTGCTG CAATGATACC GCGAGACCCA CGCTCACCGG 2250CTCCAGATTT ATCAGCAATA AACCAGCCAG CCGGAAGGGC CGAGCGCAGA 2300AGTGGTCCTG CAACTTTATC CGCCTCCATC CAGTCTATTA ATTGTTGCCG 2350GGAAGCTAGA GTAAGTAGTT CGCCAGTTAA TAGTTTGCGC AACGTTGTTG 2400CCATTGCTGC AGGCATCGTG GTGTCACGCT CGTCGTTTGG TATGGCTTCA 2450TTCAGCTCCG GTTCCCAACG ATCAAGGCGA GTTACATGAT CCCCCATGTT 2500GTGCAAAAAA GCGGTTAGCT CCTTCGGTCC TCCGATCGTT GTCAGAAGTA 2550AGTTGGCCGC AGTGTTATCA CTCATGGTTA TGGCAGCACT GCATAATTCT 2600CTTACTGTCA TGCCATCCGT AAGATGCTTT TCTGTGACTG GTGAGTACTC 2650AACCAAGTCA TTCTGAGAAT AGTGTATGCG GCGACCGAGT TGCTCTTGCC 2700CGGCGTCAAC ACGGGATAAT ACCGCGCCAC ATAGCAGAAC TTTAAAAGTG 2750CTCATCATTG GAAAACGTTC TTCGGGGCGA AAACTCTCAA GGATCTTACC 2800GCTGTTGAGA TCCAGTTCGA TGTAACCCAC TCGTGCACCC AACTGATCTT 2850CAGCATCTTT TACTTTCACC AGCGTTTCTG GGTGAGCAAA AACAGGAAGG 2900CAAAATGCCG CAAAAAAGGG AATAAGGGCG ACACGGAAAT GTTGAATACT 2950CATACTCTTC CTTTTTCAAT ATTATTGAAG CATTTATCAG GGTTATTGTC  3000TCATGAGCGG ATACATATTT GAATGTATTT AGAAAAATAA ACAAATAGGG 3050GTTCCGCGCA CATTTCCCCG AAAAGTGCCA CCTGACGTCT AAGAAACCAT 3100TATTATCATG ACATTAACCT ATAAAAATAG GCGTATCACG AGGCCCTTTC 3150 GTCTTCAAG3159 pCK7-96 (SEQ ID NO: 39)TCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGACCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATACTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGCCAAGCTAGCGGCCGCGGTCCAACCACCAATCTCAAAGCTTGGTACCCGGGAGCCTGTTATCCCAGCACAGTCCTGGAAGAGGCACAGGGGAAATAAAAGCGGACGGAGGCTTTCCTTGACTCAGCCGCTGCCTGGTCTTCTTCAGACCTGTTCTGAATTCTAAACTCTGAGGGGGTCGGATGACGTGGCCATTCTTTGCCTAAAGCATTGAGTTTACTGCAAGGTCAGAAAAGCATGCAAAGCCCTCAGAATGGCTGCAAAGAGCTCCAACAAAACAATTTAGAACTTTATTAAGGAATAGGGGGAAGCTAGGAAGAAACTCAAAACATCAAGATTTTAAATACGCTTCTTGGTCTCCTTGCTATAATTATCTGGGATAAGCATGCTGTTTTCTGTCTGTCCCTAACATGCCCTGTGATTATCCGCAAACAACACACCCAAGGGCAGAACTTTGTTACTTAAACACCATCCTGTTTGCTTCTTTCCTCAGGAACTGTGGCTGCACCATCTGTOTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGAGGGAGAAGTGCCCCCACCTGCTCCTCAGTTCCAGCCTGACCCCCTCCCATCCTTTGGCCTCTGACCCTTTTTCCACAGGGGACCTACCCCTATTGCGGTCCTCCAGCTCATCTTTCACCTCACCCCCCTCCTCCTCCTTGGCTTTAATTATGCTAATGTTGGAGGAGAATGAATAAATAAAGTGAATCTTTGCACCTGTGGTTTCTCTCTTTCCTCAATTTAATAATTATTATCTGTTGTTTACCAACTACTCAATTTCTCTTATAAGGGACTAAATATGTAGTCATCCTAAGGCGCATAACCATTTATAAAAATCATCCTTCATTCTATTTTACCCTATCATCCTCTGCAAGACAGTCCTCCCTCAAACCCACAAGCCTTCTGTCCTCACAGTCCCCTGGGCCATGGATCCTCACATCCCAATCCGCGGCCGCAATTCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGC pCG7-96 (SEQ ID NO: 40)GAACTCGAGCAGCTGAAGCTTTCTGGGGCAGGCCAGGCCTGACCTTGGCTTTGGGGCAGGGAGGGGGCTAAGGTGAGGCAGGTGGCGCCAGCCAGGTGCACACCCAATGCCCATGAGCCCAGACACTGGACGCTGAACCTCGCCGACAGTTAAGAACCCAGGGGCCTCTGCGCCCTGGGCCCAGCTCTGTCCCACACCGCGGTCACATGGCACCACCTCTCTTGCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGGTGAGAGGCCAGCACAGGGAGGGAGGGTGTCTGCTGGAAGCCAGGCTCAGCGCTCCTGCCTGGACGCATCCCGGCTATGCAGCCCCAGTCCAGGGCAGCAAGGCAGGCCCCGTCTGCCTCTTCACCCGGAGGCCTCTGCCCGCCCCACTCATGCTCAGGGAGAGGGTCTTCTGGCTTTTTCCCCAGGCTCTGGGCAGGCACAGGCTAGGTGCCCCTAACCCAGGCCCTGCACACAAAGGGGCAGGTGCTGGGCTCAGACCTGCCAAGAGCCATATCCGGGAGGACCCTGCCCCTGACCTAAGCCCACCCCAAAGGCCAAACTCTCCACTCCCTCAGCTCGGACACCTTCTCTCCTCCCAGATTCCAGTAACTCCCAATCTTCTCTCTGCAGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGGTAAGCCAGCCCAGGCCTCGCCCTCCAGCTCAAGGCGGGACAGGTGCCCTAGAGTAGCCTGCATCCAGGGACAGGCCCCAGCCGGGTGCTGACACGTCCACCTCGATCTCTTCCTCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGTGGGACCCGTGGGGTGCGAGGGCCACATGGACAGAGGCCGGCTCGGCCCACCCTCTGCCCTGAGAGTGACCGCTGTACCAACCTCTGTCCCTACAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTCGAGTOGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTGCGACGGCCGGCAAGCCCCCGCTCCCCGGGCTCTCGCGGTCGCACGAGGATGCTTGGCACGTACCCCCTGTACATACTTCCCGGGCGCCCAGCATGGAAATAAAGCACCCAGCGCTGCCCTGGGCCCCTGCGAGACTGTGATGGTTCTTTCCACGGGTCAGGCCGAGTCTGAGGCCTGAGTGGCATGAGGGAGGCAGAGCGGGTCCCACTGTCCCCACACTGGCCCAGGCTGTGCAGGTGTGCCTGGGCCCCCTAGGGTGGGGCTCAGCCAGGGGCTGCCCTCGGCAGGGTGGGGGATTTGCCAGCGTGGCCCTCCCTCCAGCAGCACCTGCCCTCGGCTGGGCCACGGGAAGCCCTAGGAGCCCCTGGGGACAGACACACAGCCCCTGCCTCTGTAGGAGACTGTCCTGTTCTGTGAGCGCCCCTGTCCTCCCGACCTCCATGCCCACTCGGGGGCATGCCTGCAGGTCGACTCTAGAGGATCCCCGGGTACCGAGCTCGAATTCATCGATGATATCAGATCTGCCGGTCTCCCTATAGTGAGTCGTATTAATTTCGATAAGCCAGGTTAACCTGGATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCGCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAACCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTAGACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTOTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATOCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGGACATATTGTCGTTAGAACGCGGCTACAATTAATACATAACCTTATGTATCATACACATACGATTTAGGTGACACTATA pG4HE (SEQ ID NO: 41)GAACTCGAGCAGCTGAAGCTTTCTGGGGCAGGCCGGGCCTGACTTTGGCTGGGGGCAGGGAGGGGGCTAAGGTGACGCAGGTGGCGCCAGCCAGGTGCACACCCAATGCCCATGAGCCCAGACACTGGACCCTGCATGGACCATCGCGGATAGACAAGAACCGAGGGGCCTCTGCGCCCTGGGCCCAGCTCTGTCCCACACCGCGGTCACATGGCACCACCTCTCTTGCAGCTTCCACCAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCCGTGACCGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTOCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGGTGAGAGGCCAGCACAGGGAGGGAGGGTGTCTGCTGGAAGCCAGGCTCAGCCCTCCTGCCTGGACGCACCCCGGCTGTGCAGCCCCAGCCCAGGGCAGCAAGGCATGCCCCATCTGTCTCCTCACCCGGAGGCCTCTGACCACCCCACTCATGCTCAGGGAGAGGGTCTTCTGGATTTTTCCACCAGGCTCCGGGCAGCCACAGGCTGGATGCCCCTACCCCAGGCCCTGCGCATACAGGGGCAGGTGCTGCGCTCAGACCTOCCAAGAGCCATATCCGGGAGGACCCTGCCCCTGACCTAAGCCCACCCCAAAGGCCAAACTCTCCACTCCCTCAGCTCAGACACCTTCTCTCCTCCCAGATCTGAGTAACTCCCAATCTTCTCTCTGCAGAGTCCAAATATGGTCCCCCATGCCCATCATGCCCAGGTAAGCCAACCCAGCCCTCGCCCTCCAGCTCAAGGCGGGACAGGTGCCCTAGAGTAGCCTGCATCCAGGGACAGGCCCCAGCCGGGTGCTGACGCATCCACCTCCATCTCTTCCTCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTCTGGTCAECGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGTGGGACCCACGGGGTGCGAGGGCCACATGGACAGAGGTCAGCTCGGCCCACCCTCTGCCCTGGGAGTGACCGCTGTGCCAACCTCTGTCCCTACAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAATGAGTGCCAGGGCCGGCAAGCCCCCGCTCCCCGGGCTCTCGGGGTCGCGCGAGGATGCTTGGCACGTACCCCGTCTACATACTTCCCAGGCACCCAGCATGGAAATAAAGCACCCACCACTGCCCTGGGCCCCTGTGAGACTGTGATGGTTCTTTCCACGGGTCAGGCCGAGTCTGAGGCCTGAGTGACATGAGGGAGGCAGAGCGGGTCCCACTGTCCCCACACTGGCCCAGGCTGTGCAGGTGTGCCTGCGCCACCTAGGGTGGGGCTCAGCCAGGGGCTGCCCTCGGCAGGGTGGGGGATTTGCCAGCGTGGCCCTCCCTCCAGCAGCAGCTGCCCTGGGCTGGGCCACGGGAAGCCCTAGGAGCCCCTGGGGACAGACACACAGCCCCTGCCTCTGTAGGAGACTGTCCTGTCCTGTGAGCGCCCTGTCCTCCGACCCCCCATGCCCACTCGGGGGGATCCCCGGGTACCGAGCTCGAATTCATCGATGATATCAGATCTGCCGGTCTCCCTATACTGACTCGTATTAATTTCGATAAGCCAGGTTAACCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAACTATATATGAGTAAACTTGGTCTGACAGTTACCAATGOTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCCACACCCACGCTCACCCGCTCCACATTTATCACCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCCCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCACTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCCTTTGGTATCGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGOCAGCACTGCATAATTCTCTTACTGTCATCCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTCCTOTTGCCCGGCGTCAATACGGGATAATACCCCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAACCGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATCACCGGATACATATTTGAATCTATTTAGAAAAATAAACAAATACCGOTTCCGCCCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCCGTCATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATCCCCGGAGCAGACAAGCCCCTCACCGCCCGTCACCGGCTGTTGGCGGCTGTCCCGGCTCCCTTAACTATGCGGCATCAGACCAGATTGTACTGAGAGTGCACCATATGGACATATTGTCGTTAGAACGCGGCTACAATTAATACATAACCTTATGTATCATACACATACGATTTAGGTGACACTATA10D1 VH (SEQ ID NO: 16)CAGGTGCAGC TGGTGGAGTC TGGGGGAGGC GTGGTCCAGC CTGGGAGGTC  50CCTGAGACTC TCCTGTGCAG CCTCTGCATT CACCTTCAGT AGCTATACTA 100TGCACTGGGT CCGCCAGGCT CCAGGCAAGG GGCTGGAGTG GGTGACATTT 150ATATCATATG ATGGAAACAA TAAATACTAC GCAGACTCCG TGAAGGGCCG 200ATTCACCATC TCCAGAGACA ATTCCAAGAA CACGCTGTAT CTGCAAATGA 250ACAGCCTGAG AGCTGAGGAC ACGGCTATAT ATTACTGTGC GAGGACCGGC 300TGGCTGGGGC CCTTTGACTA CTGGGGCCAG GGAACCCTGG TCACCGTCTC 350 CTCAG10D1 VK (SEQ ID NO: 6)GAAATTGTGT TGACGCAGTC TCCAGGCACC CTGTCTTTGT CTCCAGGGGA  50AAGAGCCACC CTCTCCTGCA GGGCCAGTCA GAGTGTTGGC AGCAGCTACT 100TAGCCTGGTA CCAGCAGAAA CCTGGCCAGG CTCCCAGGCT CCTCATCTAT 150GGTGCATTCA GCAGGGCCAC TGGCATCCCA GACAGGTTCA GTGGCAGTGG 200GTCTGGGACA GACTTCACTC TCACCATCAG CAGACTGGAG CCTGAAGATT 250TTGCAGTGTA TTACTGTCAG CAGTATGGTA GCTCACCGTG GACGTTCGGC 300CAAGGGACCA AGGTGGAAAT CAAAC 325 4B6 VH (SEQ ID NO: 18)CAGGTGCAGC TGGTGGAGTC TGGGGGAGGC GTGGTCCAGC CTGGGAGGTC  50CCTGAGACTC TCCTGTGCAG CCTCTGGATT CACCTTCAGT AGCTATACTA 100TGCACTGGGT CCGCCAGGCT CCAGGCAAGG GGCTGGAGTG GGTGACATTT 150ATATCATATG ATGGAAGCAA TAAACACTAC GCAGACTCCG TCAACCGCCG 200ATTCACCGTC TCCAGAGACA ATTCCAAGAA CACGCTGTAT CTGCAAATGA 250ACAGCCTGAG AGCTGAGGAC ACGGCTATAT ATTACTGTGC GAGGACCGGC 300TGGCTGGGGC CCTTTGACTA CTGGGGCCAG GGAACCCTGG TCACCGTCTC 350 CTCAG4B6 VK (SEQ ID NO: 8)GAAATTGTGT TGACGCAGTC TCCAGGCACC CTGTCTTTGT CTCCAGGGGA  50AAGAGCCACC CTCTCCTGCA GGGCCAGTCA GAGTGTTAGC AGCAGCTTCT 100TAGCCTGGTA CCAGCAGAAA CCTGGCCAGG CTCCCAGGCT CCTCATCTAT 150GGTGCATCCA GCAGGGCCAC TGGCATCCCA GACAGGTTCA GTGGCAGTGG 200GTCTGGGACA GACTTCACTC TCACCATCAG CAGACTGGAG CCTGAAGATT 250TTGCAGTGTA TTACTGTCAG CAGTATGGTA GCTCACCGTG GACGTTCGGC 300CAAGGGACCA AGGTGGAAAT CAAAC 325 1E2 VH (SEQ ID NO: 22)CAGGTGCAGC TGGTGGAGTC TGGGGGAGGC GTGGTCCAGC CTGGGAGGTC  50CCTGAGACTC TCCTGTGCAG CGTCTGGATT CACCTTCAGT AGCTATOGCA 100TGCACTGGGT CCGCCAGGCT CCTGGCCAGG GGCTGGAGTG GGTGGCAGTT 150ATATGGTATG ATGGAAGTAA TAAATACTAT GCAGACTCCG TGAAGGGCCG 200ATTCACCATC TCCAGAGACA ATTCCAAGAA CACGCTGTAT CTGCAAATGA 250ACAGCCTGAG AGCCGAGGAC ACGGCTGTGT TTTACTGTGC GAGAGCTCCC 300AATTATATTG GTGCTTTTGA TGTCTGGGGC CAAGGGACAA TGGTCACCGT 350 CTCTTCAG1E2 VK (SEQ ID NO: 12)GACATCCAGA TGACCCAGTC TCCATCCTCA CTGTCTGCAT CTGTAGGAGA  50CAGAGTCACC ATCACTTGTC GGGCGAGTCA GGGTATTAGC AGCTGGTTAG 100CCTGGTATCA GCAGAAACCA GAGAAAGCCC CTAAGTCCCT GATCTATGCT 150GCATCCAGTT TGCAAAGTGG GGTCCCATCA AGGTTCAGCG GCAGTGGATC 200TGGGACAGAT TTCACTCTCA CCATCAGCAG CCTGCAGCCT GAAGATTTTG 250CAACTTATTA CTGCCAACAG TATAATAGTT ACCCTCCGAC GTTCGGCCAA 300GGGACCAAGG TGGAAATCAA AC 322

What is claimed is:
 1. A method for increasing an immune response to anantigen in a subject, the method comprising administering to the subjectan anti-CTLA-4 antibody or antigen-binding portion thereof, wherein theantibody or antigen-binding portion thereof comprises a heavy chainvariable region that comprises CDR1, CDR2, and CDR3 domains; and a lightchain variable region that comprises CDR1, CDR2, and CDR3 domains,wherein the heavy chain variable region and light chain variable regionCDR3 domains are selected from the group consisting of: (a) a heavychain variable region CDR3 comprising amino acids having the sequenceset forth in SEQ ID NO:37; and a light chain variable region CDR3comprising amino acids having the sequence set forth in SEQ ID NO:35;and (b) a heavy chain variable region CDR3 comprising amino acids havingthe sequence set forth in SEQ ID NO:38; and a light chain variableregion CDR3 comprising amino acids having the sequence set forth in SEQID NO:36; and binds to human CTLA-4 with a binding affinity of about 10⁸M⁻¹ or greater.
 2. The method of claim 1, wherein the antibody orantigen-binding portion thereof comprises: (a) a heavy chain variableregion comprising CDR3 and CDR2 sequences set forth in SEQ ID NOs:37 and32, respectively; and a light chain variable region comprising CDR3 andCDR2 sequences set forth in SEQ ID NOs:35 and 29, respectively; (b) aheavy chain variable region comprising CDR3 and CDR2 sequences set forthin SEQ ID NOs: 37 and 33, respectively; and a light chain variableregion comprising CDR3 and CDR2 sequences set forth in SEQ ID NOs: 35and 30, respectively; or (c) a heavy chain variable region comprisingCDR3 and CDR2 sequences set forth in SEQ ID NOs:38 and 34, respectively;and a light chain variable region comprising CDR3 and CDR2 sequences setforth in SEQ ID NOs:36 and 31, respectively.
 3. The method of claim 2,wherein the antibody or antigen-binding portion thereof comprises: (a) aheavy chain variable region comprising CDR3, CDR2 and CDR1 sequences setforth in SEQ ID NOs:37, 32 and 27, respectively; and a light chainvariable region comprising CDR3, CDR2 and CDR1 sequences set forth inSEQ ID NOs:35, 29 and 24, respectively; (b) a heavy chain variableregion comprising CDR3, CDR2 and CDR1 sequences set forth in SEQ IDNOs:37, 33 and 27, respectively; and a light chain variable regioncomprising CDR3, CDR2 and CDR1 sequences set forth in SEQ ID NOs:35, 30and 25, respectively; or (c) a heavy chain variable region comprisingCDR3, CDR2 and CDR1 comprising sequences set forth in SEQ ID NOs:38, 34and 28, respectively; and a light chain variable region comprising CDR3,CDR2 and CDR1 sequences set forth in SEQ ID NOs:36, 31 and 26,respectively.
 4. The method of claim 1, wherein the antibody orantigen-binding portion thereof comprises: (a) a heavy chain variableregion derived from a human V_(H) 3-30.3 gene; and (b) a light chainvariable region derived from a human V_(K) A-27 gene.
 5. The method ofclaim 1, wherein the antibody or antigen-binding portion thereofcomprises: (a) a heavy chain variable region derived from a human V_(H)3-33 gene; and (b) a light chain variable region derived from a humanV_(K) L-15 gene.
 6. The method of claim 4, wherein the antibody orantigen-binding portion thereof comprises a heavy chain variable regionCDR3 sequence set forth in SEQ ID NO: 37, and a light chain variableregion CDR3 sequence set forth in SEQ ID NO:
 35. 7. The method of claim5, wherein the antibody or antigen-binding portion thereof comprises aheavy chain variable region CDR3 sequence set forth in SEQ ID NO: 38,and a light chain variable region CDR3 sequence set forth in SEQ ID NO:36.
 8. The method of claim 1, wherein the antibody or antigen-bindingportion thereof comprises at least one heavy chain variable regioncomprising an amino acid sequence selected from the group consisting ofSEQ ID NOS: 17, 19 and
 23. 9. The method of claim 1, wherein theantibody or antigen-binding portion thereof comprises at least one lightchain variable region comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOS: 7, 9 and
 13. 10. The method of claim1, wherein the antibody or antigen-binding portion thereof comprises aheavy chain variable region comprising the amino acid sequence set forthin SEQ ID NO: 17 and a light chain variable region comprising the aminoacid sequence set forth in SEQ ID NO:
 7. 11. The method of claim 1,wherein the antibody or antigen-binding portion thereof comprises aheavy chain variable region comprising the amino acid sequence set forthin SEQ ID NO: 19 and a light chain variable region comprising the aminoacid sequence set forth in SEQ ID NO:
 9. 12. The method of claim 1,wherein the antibody or antigen-binding portion thereof comprises aheavy chain variable region comprising the amino acid sequence set forthin SEQ ID NO: 23 and a light chain variable region comprising the aminoacid sequence set forth in SEQ ID NO:
 13. 13. The method of claim 1,wherein the antibody or antigen-binding portion thereof comprises: (a) aheavy chain variable region comprising CDR1, CDR2, and CDR3 sequencesset forth in SEQ ID NOS: 27, 32 and 37, respectively; and (b) a lightchain variable region comprising CDR1, CDR2, and CDR3 sequences setforth in SEQ ID NOS: 24, 29 and 35, respectively.
 14. The method ofclaim 1, wherein the antibody or antigen-binding portion thereofcomprises: (a) a heavy chain variable region comprising CDR1, CDR2, andCDR3 sequences set forth in SEQ ID NOS: 27, 33 and 37, respectively; and(b) a light chain variable region comprising CDR1, CDR2, and CDR3sequences set forth in SEQ ID NOS: 25, 30 and 35, respectively.
 15. Themethod of claim 1, wherein the antibody or antigen-binding portionthereof comprises: (a) a heavy chain variable region comprising CDR1,CDR2, and CDR3 sequences set forth in SEQ ID NOS: 28, 34 and 38,respectively; and (b) a light chain variable region comprising CDR1,CDR2, and CDR3 sequences set forth in SEQ ID NOS: 26, 31 and 36,respectively.
 16. A method for increasing an immune response to anantigen in a subject, the method comprising administering to the subjectan antibody or antigen-binding portion thereof, wherein the antibody orantigen-binding portion thereof competes for binding to a human CTLA-4polypeptide with a reference antibody comprising the amino acid sequenceset forth in SEQ ID NO: 17, and the amino acid sequence set forth in SEQID NO: 7; and has a binding affinity of about 10⁸M⁻¹ or greater.
 17. Amethod for increasing an immune response to an antigen in a subject, themethod comprising administering to the subject an anti-CTLA-4 antibodyor antigen-binding portion thereof, comprising: (a) a heavy chainvariable region comprising CDR1, CDR2, and CDR3 sequences set forth inSEQ ID NOS: 27, 32 and 37, respectively; and (b) a light chain variableregion comprising CDR1, CDR2, and CDR3 sequences set forth in SEQ IDNOS: 24, 29 and 35, respectively.
 18. A method for increasing an immuneresponse to an antigen in a subject, the method comprising administeringto the subject an anti-CTLA-4 antibody or antigen-binding portionthereof, comprising: (a) a heavy chain variable region comprising CDR1,CDR2, and CDR3 sequences set forth in SEQ ID NOS: 27, 33 and 37,respectively; and (b) a light chain variable region comprising CDR1,CDR2, and CDR3 sequences set forth in SEQ ID NOS: 25, 30 and 35,respectively.
 19. A method for increasing an immune response to anantigen in a subject, the method comprising administering to the subjectan anti-CTLA-4 antibody or antigen-binding portion thereof, comprising:(a) a heavy chain variable region comprising CDR1, CDR2, and CDR3sequences set forth in SEQ ID NOS: 28, 34 and 38, respectively; and (b)a light chain variable region comprising CDR1, CDR2, and CDR3 sequencesset forth in SEQ ID NOS: 26, 31 and 36, respectively.
 20. A method forincreasing an immune response to an antigen in a subject, the methodcomprising administering to the subject an anti-CTLA-4 antibody orantigen-binding portion thereof comprising a heavy chain variable regioncomprising the amino acid sequence set forth in SEQ ID NO: 17 and alight chain variable region comprising the amino acid sequence set forthin SEQ ID NO:
 7. 21. A method for increasing an immune response to anantigen in a subject, the method comprising administering to the subjectan anti-CTLA-4 antibody or antigen-binding portion thereof comprising aheavy chain variable region comprising the amino acid sequence set forthin SEQ ID NO: 19 and a light chain variable region comprising the aminoacid sequence set forth in SEQ ID NO:
 9. 22. A method for increasing animmune response to an antigen in a subject, the method comprisingadministering to the subject an anti-CTLA-4 antibody or antigen-bindingportion thereof comprising a heavy chain variable region comprising theamino acid sequence set forth in SEQ ID NO: 23 and a light chainvariable region comprising the amino acid sequence set forth in SEQ IDNO:
 13. 23. The method of any one of claims 1 and 16-22, wherein theantibody is a human antibody.
 24. The method of any one of claims 1 and16-22, wherein the antibody is a monoclonal antibody.
 25. The method ofany one of claims 1 and 16-22, wherein the antibody or antigen-bindingportion thereof inhibits binding of human CTLA-4 to B7-1 or B7-2. 26.The method of any one of claims 1 and 16-22, wherein the antigen is atumor antigen.
 27. The method of claim 26, wherein the tumor antigen isselected from the group consisting of a prostate tumor antigen, amelanoma tumor antigen, an epithelial tumor antigen, and a combinationthereof.
 28. The method of any one of claims 1 and 16-22, furthercomprising administering an antigen, or a fragment or an analog thereof,to the subject, wherein administration of the antigen in combinationwith the antibody, or antigen-binding portion thereof, increases theimmune response to the antigen.
 29. The method of claim 28, wherein theantigen is a tumor antigen.